1. Fahimtar farko

A wannan mataki, muna buƙatar fahimtar wasu ra'ayoyi da kalmomi, don guje wa yin kuskure a gaban manyanmu, kamar:

Tambaya: Menene bambanci tsakanin RT-PCR, qPCR, PCR na ainihi, da kuma RT-PCR na ainihi?

Amsa: RT-PCR ita ce juyar da rubutun PCR(Reverse transcription PCR, RT-PCR), wanda shine bambance-bambancen sarkar polymerase (PCR).A cikin RT-PCR, ana jujjuya madaidaicin RNA zuwa DNA na ƙarin, wanda PCR ke amfani dashi azaman samfuri don haɓaka DNA.
Real-lokaci-PCR da qPCR(Quantitative Rea-ltime-PCR) abu ɗaya ne, duka biyun PCR masu ƙididdigewa ne na ainihin lokaci, wanda ke nufin cewa kowane zagayowar PCR yana da bayanan bayanan lokaci na ainihi, don haka ana iya daidaita adadin samfuran farawa daidai bincike.

Ko da yake duka PCR na lokaci-lokaci (ainihin ƙimar ƙimar PCR) da kuma Reverse transcription PCR (PCR na juyi) da alama ana rage su azaman RT-PCR, yarjejeniyar kasa da kasa ita ce: RT-PCR musamman tana nufin juyawa juzu'i.PCR , PCR na ainihi ana taƙaita shi azaman qPCR (ƙirar PCR na ainihin lokaci).

Kuma RT-PCR na ainihi (RT-qPCR), PCR ce ta juyar da aka haɗa tare da fasahar ƙididdigewa mai kyalli.: da farko sami cDNA (RT) daga rubutun juzu'i na RNA, sannan a yi amfani da PCR na ainihi don ƙididdigar ƙididdigewa (qPCR).Yawancin dakunan gwaje-gwaje suna yin RT-qPCR, wato, bincike kan ƙa'idar bayyanar RNA, don haka qPCR da kowa yayi magana game da shi a cikin dakin gwaje-gwaje a zahiri yana nufin RT-qPCR, amma kar a manta cewa har yanzu akwai gwajin DNA da yawa a aikace-aikacen asibiti.Ƙididdigar ƙididdigewa, irin su cutar hanta ta HBV.

Tambaya: Bayan karanta yawancin PCR mai kyalli, me yasa za'a sarrafa guntuwar da aka ƙara a cikin kewayon 80-300bp?

Amsa: Tsawon kowane jerin kwayoyin halitta ya bambanta, wasu suna da kb da yawa, wasu daruruwan bp ne, amma muna buƙatar kawai buƙatar tsawon samfurin ya zama 80-300bp lokacin zayyana abubuwan ƙira, gajere ko tsayi da yawa ba su dace da gano PCR mai kyalli ba.Rukunin samfurin ya yi gajere da yawa don a bambanta shi da madaidaicin dimer.Tsawon dimer-dimer yana kusan 30-40bp, kuma yana da wuya a rarrabe ko yana da dimer-dimer ko samfur idan bai wuce 80bp ba.Idan guntun samfurin ya yi tsayi da yawa, ya wuce 300bp, zai iya haifar da ƙarancin haɓakawa cikin sauƙi kuma ba zai iya gano adadin kwayoyin halitta yadda ya kamata ba.

Misali, idan ka kirga mutane nawa ne a cikin aji, kawai kuna bukatar kirga bakunan nawa ne.Haka abin yake idan kun gano kwayoyin halitta, kawai kuna buƙatar gano wani jerin jerin kwayoyin halitta don wakiltar Duk jerin za su yi.Idan kana so ka ƙidaya mutane, kana buƙatar ƙidaya baki da hanci, kunnuwa, da tabarau, kuma yana da sauƙi a yi kuskure.

Don faɗaɗa, a cikin binciken nazarin halittu, akwai lokuta masu yawa na bincike daga aya zuwa yanki, saboda jerin kwayoyin halittar kowane nau'in suna da tsayi sosai, ba dole ba ne kuma ba zai yiwu a auna dukkan gutsuttsura ba, kamar kwayoyin 16S sequencing, wanda shine aiwatar da tsarin masu ra'ayin mazan jiya na Bacteria Assays don ƙididdige adadin adadin adadin yawan adadin kwayoyin cutar.

Tambaya: Menene mafi kyawun tsayi don ƙirar ƙirar qPCR?

Amsa: Gabaɗaya magana, tsayin farko shine kusan 20-24bp, wanda shine mafi kyau.Tabbas, dole ne mu mai da hankali ga ƙimar TM na firamare yayin zayyana firam ɗin, saboda wannan yana da alaƙa da mafi kyawun zafin jiki na annealing.Bayan gwaje-gwaje da yawa, an tabbatar da cewa 60 ° C shine mafi kyawun ƙimar TM.Idan zafin zafin da ke rufewa ya yi ƙasa da ƙasa, zai iya haifar da haɓakawa maras takamaiman.Idan yawan zafin jiki na annealing ya yi yawa, ingancin haɓakawa zai yi ƙasa kaɗan, kololuwar ƙarawar ƙarawa zai fara daga baya, kuma ƙimar CT za ta jinkirta.

Tambaya: Ta yaya hanyar rini ta bambanta da hanyar bincike?

Amsa: Hanyar RiniWasu rini mai kyalli, irin su SYBR Green Ⅰ, PicoGreen, BEBO, da sauransu, ba sa fitar da haske da kansu, amma za su fitar da haske bayan sun ɗaure ga ƙaramin tsagi na DNA mai ɗaure biyu.Sabili da haka, a farkon amsawar PCR, injin ba zai iya gano siginar kyalli ba.Lokacin da abin ya kai matakin tsawa-tsawa, za a buɗe igiya biyu, kuma an haɗa sabon igiya a ƙarƙashin aikin DNA polymerase, kuma ƙwayar kyalli yana ɗaure zuwa ƙaramin tsagi na dsDNA.Yayin da adadin zagayowar PCR ke ƙaruwa, ana ƙara yawan rini da ake haɗawa tare da DNA mai ɗaci biyu, kuma siginar kyalli shima yana ci gaba da haɓakawa.Ana amfani da hanyar rini galibi a cikin binciken kimiyya.
PS: Yi hankali lokacin yin gwajin, dole ne a haɗa rini tare da DNA na ɗan adam, a kula don juya shi zuwa mutum mai kyalli.

Hanyar rini (hagu) Hanyar bincike (dama)
PS: Yi hankali lokacin yin gwajin, dole ne a haɗa rini tare da DNA na ɗan adam, a kula don juya shi zuwa mutum mai kyalli.

SYBR Green Ⅰ yana ɗaure ga ƙaramin tsagi na DNA

Hanyar bincikeBinciken Taqman shine binciken hydrolysis da aka fi amfani dashi.Akwai rukuni mai kyalli a ƙarshen 5′ na binciken, yawanci FAM, kuma binciken da kansa jeri ne wanda ya dace da asalin da aka yi niyya.Akwai ƙungiyar kashe kyalli a ƙarshen 3′.Dangane da ka'idar Canjawar Canjin Canjin Canjin Canjin Canjin Canjin Canjin Canji (Förster Resonance Energy Transfer, FRET), lokacin da ƙungiyar mai ba da labari mai kyalli (mai ba da gudummawa mai kyalli) da ƙungiyar quenching fluorescent (ƙarashin ƙwayoyin kyalli) suna jin daɗi Lokacin da spectra zoba da nisa yana kusa (7-10n na iya ba da gudummawa) kwayoyin halitta, yayin da autofluorescence ya raunana.Sabili da haka, a farkon amsawar PCR, lokacin da binciken ya kasance kyauta kuma yana cikin tsarin, ƙungiyar masu ba da labari ba za ta fitar da haske ba.Lokacin da ake cirewa, firamare da bincike suna ɗaure da samfuri.A lokacin matakin tsawo, polymerase yana ci gaba da haɗa sabbin sarƙoƙi.DNA polymerase yana da 5'-3' aikin exonuclease.Lokacin isa binciken, polymerase na DNA zai ɗora ruwan bincike daga samfuri, ya ware ƙungiyar masu kyalli daga ƙungiyar masu kyalli, sannan ta saki siginar kyalli.Tun da akwai alaƙa ɗaya zuwa ɗaya tsakanin binciken da samfuri, hanyar binciken ta fi hanyar rini dangane da daidaito da azancin gwajin.Ana amfani da hanyar bincike musamman wajen gano cutar.

Tambaya: Menene cikakken ƙididdigewa?Menene Ƙididdigar Ƙwararru?

AmsaCikakken ƙididdigewa yana nufin ƙididdige lambar kwafin farko na samfurin da za a gwada ta qPCR, kamar nawa ƙwayoyin cutar HBV ke cikin 1ml na jini.Sakamakon da aka samu ta hanyar ƙididdigewa na dangi shine canji a cikin adadin ƙwayar da aka yi niyya a cikin takamaiman samfurin dangi zuwa wani samfurin tunani, kuma an daidaita bayanin kwayar halitta ko an daidaita shi.

Tambaya: Shin adadin hakar RNA, da juyar da ingancin rubutun, da ingancin haɓakawa zai shafi sakamakon gwaji?
Tambaya: Shin samfurin ajiya, reagents na hakar, reagents na jujjuya rubutu, da abubuwan da ake watsa haske suna shafar sakamakon gwaji?
Tambaya: Wace hanya ce za ta iya gyara bayanan gwaji?

Game da waɗannan batutuwa, za mu bayyana su dalla-dalla a cikin ci gaba da ci gaba da sassan da ke ƙasa.
2. Nagartaccen ilimi

Dangane da PCR mai kyalli na ainihin lokaci, dole ne mu gane gaskiyar cewa ana buga dubban takaddun bincike na kimiyya kowace shekara, daga cikinsu fasahar PCR mai kyalli ba ƙaramin adadi ba ne.

Idan babu ma'auni gama gari don auna gwajin ƙimar PCR mai kyalli, sakamakon zai iya bambanta sosai.Ga kwayoyin halittar nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i) nau'i-nau'i-nau'i-nau'i-nau'i-nau'i-nau'i-nau'i.Kai Ba wanda ya san wanne ne daidai da wanda ba daidai ba.

Shin wannan yana nufin cewa PCR mai kyalli fasaha ce ta yaudara ko fasaha mara dogaro?A'a, saboda PCR mai kyalli ya fi hankali kuma ya fi daidai, kuma ɗan ƙaramin aiki ba daidai ba zai haifar da sakamako gaba ɗaya.Karamar asara tana da nisa mil dubu.Marubucin labarin na iya fuskantar azabtarwa akai-akai ta masu bitar.A lokaci guda kuma, masu bitar mujallar suna da wahalar zaɓar daga sakamakon gwaji daban-daban.

Gabaɗaya, yana nuna rashin daidaituwa a cikin gwaje-gwajen PCR na ainihi.Don haka, manyan masana kimiyya a masana'antar sun fara tsara ma'auni,yana buƙatar masu ba da gudummawa don samar da wasu mahimman bayanan gwaji da sarrafa bayanai (ciki har da mahimman bayanai) a cikin labarin don saduwa da waɗannan ƙa'idodi.

Masu dubawa za su iya yin la'akari da ingancin gwajin ta hanyar karanta waɗannan cikakkun bayanai;Masu karatu na gaba kuma za su iya amfani da wannan don maimaita gwajin ko inganta gwajin.Sa'an nan sakamakon gwajin da aka samu ta wannan hanya yana cike da bayanai, masu inganci, kuma masu amfani.

MIBBI (Ƙaramar Bayani don Binciken Halittu da Kwayoyin Halitta -http://www.mibbi.org) ya kasance.MIBBI aiki ne da ke ba da ma'auni don gwaji.An buga shi a cikin yanayi.Wannan aikin yana nufin gwaje-gwajen halittu daban-daban, gami da ilmin halitta cell, Microarray, qPCR da za mu tattauna a yanzu, da sauransu, kuma yana ba da kowane nau'in gwaji yayin ƙaddamar da rubutun hannu.Ya kamata a ba da wannan bayanin a kowane lokaci.

A cikin aikin MIBBI, akwai labarai guda biyu masu alaƙa da PCR mai ƙima, wato:
RDML (Harshen Alamar Bayanan Bayanai na PCR na ainihi) - ingantaccen harshe da jagorar bayar da rahoto don ƙimar PCR na ainihin lokaci;
· MIQE (Ƙaramar Bayani don Buga Gwaje-gwajen PCR na Gaskiya na Gaskiya) - ƙaramin bayani don buga labarai game da gwaje-gwajen PCR na ainihin-lokaci.
Da farko, bari muyi magana game da RDML, ƙayyadaddun kalmomi.

Idan babu daidaitattun ma'anar komai, ba shi yiwuwa a ci gaba da tattaunawa, wanda shine dalilin da ya sa bayanin sharuɗɗan yana da mahimmanci a cikin jarrabawa.
Kalmomin da aka yi amfani da su a gwajin PCR mai kyalli ya haɗa da abun ciki mai zuwa.QIAGEN ya yi mana mafi kyawun taƙaitawa.Wadannan duk sun bushekaya .

Lanƙwan ƙarawa
Ƙwaƙwalwar haɓakawa tana nufin lanƙwan da aka yi yayin aiwatar da PCR, tare da lambar sake zagayowar azaman abscissa da ainihin lokacin haske mai haske yayin amsawa a matsayin ordinate.

Kyakkyawan madaidaicin ƙarawa yakamata ya sami halaye masu zuwa: tushen tushe yana da lebur ko an rage kadan, kuma babu wani yanayi na sama a bayyane;madaidaicin juzu'i na lanƙwasa a bayyane yake, kuma gangara na lokaci mai faɗi daidai yake da ingancin haɓakawa.Mafi girman gangaren, mafi girman ingancin haɓakawa;madaidaicin ƙararrawa gabaɗaya Daidaituwa yana da kyau, yana nuna cewa haɓakar haɓakar kowane bututu yana kama da haka;ɓangarorin ɓangarorin ƙaƙƙarfan ƙaƙƙarfan samfuran ƙima a bayyane yake.

Baseline (Baseline)
Tushen shine matakin amo na farkon zagayowar, yawanci ana aunawa tsakanin zagayowar 3rd da 15th, saboda haɓakar ƙimar kyalli wanda samfurin ƙarawa ya haifar ba za a iya gano shi ba a wannan lokacin.Adadin zagayowar da aka yi amfani da shi don ƙididdige tushen tushe na iya bambanta kuma yana iya buƙatar ragewa idan an yi amfani da adadin samfuri masu yawa ko kuma idan matakin magana na jigon manufa ya yi girma.

Kafa tushen tushe yana buƙatar duba bayanan haske daga layin ƙara girman layi.An saita tushen tushe don haɓakar juzu'in haɓakawa ya fara da lambar zagayowar mafi girma fiye da lambar saman zagayowar tushe.Ana buƙatar saita tushen tushe guda ɗaya don kowane jerin manufa.Matsakaicin ƙimar haske da aka gano a farkon zagayowar suna buƙatar cirewa daga ƙimar haske da aka samu a cikin haɓakar samfuran.Sabbin nau'ikan software na PCR na Real-Time daban-daban suna ba da damar haɓaka saitunan asali ta atomatik don samfuran mutum ɗaya.

A lokacin ƴan zagayowar farko na amsawar haɓakawa na PCR, siginar kyalli ba ya canzawa da yawa.Kusanci madaidaicin layi ana kiransa tushe, amma idan muka duba da kyau a ƴan zagayowar farko, zamu ga cewa a cikin tushe shine abin da ke faruwa a hoton da ke ƙasa.

Bayan Fage yana nufin
Ƙimar haske mara ƙayyadadden ƙayyadaddun abu a cikin dauki .Misali: rashin inganci quenching fluorescence;ko adadi mai yawa na samfuran DNA mai ɗaure biyu saboda amfani da SYBR Green.Abubuwan da ke bayan siginar ana cire su ta hanyar lissafi ta hanyar software na Real-Time PCR algorithm.

Alamar mai rahoto
Siginar mai ba da rahoto tana nufin siginar kyalli wanda SYBR Green ke samarwa ko takamaiman bincike-bincike na musamman a lokacin Real-Time PCR.

Siginar Mai Rahoto Na Al'ada (RN)
RN yana nufin tsananin kyalli na rini na mai ba da rahoto wanda aka raba ta wurin ƙarfin haske na rini mai wucewa da aka auna a kowane zagayowar.

Rini Mai Mahimmanci
A cikin wasu PCRs na Real-Time,Ana amfani da rini mai kyalli ROX azaman abin tunani na ciki don daidaita siginar kyalli.Yana gyara don bambance-bambancen saboda rashin daidaitaccen bututu, matsayi mai kyau, da sauye-sauyen walƙiya akan rijiyar da kyau.

Ƙofar ƙofa (ƙofa)
an daidaita shi sama da ƙimar bango kuma yana ƙasa da ƙimar faranti na lanƙwan ƙarawa.Dole ne ya kwanta a cikin layi na layin haɓakawa, yana wakiltar kewayon linzamin kwamfuta na gano PCR.Ya kamata a saita maƙasudin a cikin ra'ayi na haɓaka log-amplification ta yadda za'a iya gane lokacin log-linear na PCR cikin sauƙi.Idan akwai kwayoyin halitta da yawa na manufa a cikin PCR na Real-Time, dole ne a saita ƙofa don kowane manufa.Gabaɗaya, ana amfani da siginar kyalli na zagayowar 15 na farko na amsawar PCR azaman siginar bangon haske, kuma madaidaicin ƙyalli sau 10 daidaitaccen siginar kyalli na farkon 3 zuwa 15 hawan keke na PCR, kuma an saita madaidaicin ƙyalli a cikin lokacin girma na PCRl.Gabaɗaya, kowane kayan aiki yana da madaidaicin haske da aka saita kafin amfani.

Ƙarfin Zagaye (CT) ko Wurin Ketare (CP)
Zagayewar da lanƙwan ƙarawa ta ƙetare bakin kofa (watau wurin da gano haske ya ƙaru sosai).CT na iya zama juzu'i kuma ana iya ƙididdige adadin samfurin farawa.Ƙimar CT tana wakiltar adadin zagayowar da aka samu lokacin da siginar kyalli a cikin kowane bututun dauki na PCR ya kai matakin da aka saita.Akwai dangantaka ta layi tsakanin ƙimar CT na kowane samfuri da logarithm na lambar kwafin farko na samfurin,mafi girma lambar kwafin farko, ƙarami ƙimar CT, kuma akasin haka.Ana iya yin daidaitaccen lanƙwasa ta amfani da ma'auni tare da sanannen lambar kwafin farko, inda abscissa ke wakiltar ƙimar CT, kuma ordinate yana wakiltar logarithm na lambar kwafin farko.Sabili da haka, idan dai ana samun ƙimar CT na samfurin da ba a sani ba, ana iya ƙididdige lambar kwafin farko na samfurin daga daidaitattun layi.

ΔCT darajar
ΔCT darajar ta bayyanabambanci tsakanin jigon da aka yi niyya da madaidaicin ma'anar jigon jigon CT ƙimar, kamar kwayar halitta, kuma ana amfani da ita don daidaita yawan samfurin da aka yi amfani da shi:
⇒ΔCT = CT (jinin manufa) - CT (geneous reference gene)

ΔΔCT Darajar
Ƙimar ΔΔCT ta kwatanta bambanci tsakanin ma'anar ΔΔCT na samfurin sha'awa (misali, ƙwayoyin da aka haɓaka) da ma'anar ΔΔCT na samfurin tunani (misali, sel marasa ƙarfi).Hakanan ana kiran samfurin tunani samfurin calibration kuma duk sauran samfuran an daidaita su zuwa wannan don ƙididdige dangi:
⇒ΔΔCT = matsakaicin ΔCT (samfurin sha'awa) - matsakaicin ΔCT (samfurin nuni)

Ƙwayoyin halitta na asali (Genes reference genes)
Matakan magana na kwayoyin halitta masu mahimmanci, kamar kwayoyin halitta (jinin kula da gida), ba su bambanta tsakanin samfurori ba.Kwatanta dabi'un CT na ma'anar kwayar halitta zuwa kwayar da aka yi niyya yana ba da damar matakin magana na jigon manufa don daidaitawa zuwa adadin shigarwar RNA ko cDNA (duba sashe akan ƙimar ΔCT a sama).

Kwayoyin bincike na ciki daidai donyuwuwar lalata RNA ko kasancewar masu hana enzyme a cikin samfuran RNA, da kuma bambance-bambance a cikin abun ciki na RNA, juyar da ingancin rubutun, dawo da acid nucleic, da sarrafa samfurin.Don zaɓar mafi kyawun jigon tunani (s), mun canza algorithm don ba da damar zaɓin mafi kyawun tunani wanda ya dogara da saitin gwaji.

Ikon cikin gida
Tsarin sarrafawa wanda aka haɓaka a cikin amsa iri ɗaya da jerin manufa kuma aka bincika tare da bincike daban (watau yin PCR duplex).Sau da yawa ana amfani da sarrafawar ciki don yin watsi da abubuwan haɓakawa da suka gaza, kamar lokacin da ba a gano jerin abubuwan da aka yi niyya ba.
Samfurin daidaitawa
Samfurin tunani (misali, tsarkakewar RNA daga layin tantanin halitta ko nama) da aka yi amfani da shi cikin ƙididdigewa na dangi don kwatanta duk sauran samfuran don tantance matakin faɗin dangi na kwayar halitta.Samfurin gyare-gyare na iya zama kowane samfurin, amma yawanci shine sarrafawa (misali, samfurin da ba a kula da shi ba ko samfurin daga lokacin sifilin gwajin).

Ingantattun sarrafawa
yi amfani da halayen sarrafawa tare dasanannun adadin samfuri.Ana amfani da ingantattun sarrafawa sau da yawa don bincika cewa saitin firamare ko saitin bincike-saitin bincike yana aiki da kyau kuma an saita matakin daidai.

Babu Ikon Samfura (NTC)
Maganin sarrafawa wanda ya ƙunshi duk abubuwan da ake buƙata na haɓaka haɓakawa sai dai samfuri, wanda yawanci ana maye gurbinsa da ruwa.Amfani da NTC na iya samun gurɓataccen gurɓataccen gurɓataccen abu ko DNA na ƙasashen waje, don haka tabbatar da sahihanci da amincin bayanan ganowa.Ƙara ƙarfin sarrafa NTC yana nuna gurɓatawa.

Babu ikon sarrafa RT (NRT)
Tsarin hakar RNA na iya ƙunsar ragowar DNA na genomic, wanda ke da cutarwa sosai kuma shine mai laifi yana shafar ingancin bayanai da kuma makiyin qPCR, don haka lokacin zayyana gwaje-gwaje, dole ne a tsara shi don haɓaka gano RNA kawai.Akwai hanyoyi guda biyu, ɗaya shine don tsara abubuwan da aka tsara a cikin introns, ɗayan shine don cire DNA gaba ɗaya, wanda ya fi kyau, wanda za'a tattauna daga baya.Ikon NTR madubin sihiri ne don gano gurɓacewar DNA.Idan akwai ƙarawa, yana nufin akwai gurɓatacce.

Matsayi
Ma'auni samfuran sananniya ne na taro ko kwafin lamba waɗanda ake amfani da su don gina madaidaicin lanƙwasa.Don tabbatar da kwanciyar hankali na ma'auni, ɓangarorin kwayoyin yawanci ana cloned cikin plasmid kuma ana amfani dashi azaman ma'auni.

Madaidaicin lankwasa
yawanci ana diluted a cikin aƙalla gradients na taro 5 tare da daidaitaccen samfurin bisa ga rabo mai ninki biyu, kuma ana zana maki 5 a cikin madaidaicin ƙimar CT da lambar kwafi, kuma ana haɗa maki don samar da layi don samar da daidaitaccen lanƙwasa.Ga kowane madaidaicin kwana, ana buƙatar bincika ingancin sa.Ƙimar gangara ta faɗi tsakanin -3.3 da -3.8, kuma kowane taro ana yin shi sau uku.Ya kamata a yi watsi da abubuwan da suka bambanta da sauran maki.Ana kawo darajar CT na samfurin da za a gwada a cikin daidaitaccen ma'auni, kuma ana iya ƙididdige matakin magana na samfurin da za a gwada.

Ana kawo ƙimar CT na samfurin da za a gwada a cikin daidaitaccen lanƙwasa, kuma ana iya ƙididdige lambar kwafin farko na samfurin da za a gwada.

inganci da gangara
Matsakaicin madaidaicin lanƙwasa yana wakiltar ingancin PCR na ainihin-lokaci.
-Tsarin -3.322 yana nuna cewa ingancin haɓakawa na PCR shine 1, ko 100% inganci, kuma adadin samfuran PCR ya ninka sau biyu a kowane zagayowar.
Wani gangare ƙasa da -3.322 (misali, -3.8) yana nuna ingancin PCR
Wani gangare mai girma fiye da -3.322 (misali, -3.0) yana nuna cewa ingancin PCR ya bayyana ya fi 100%, wanda ke da sha'awar, ta yaya za a iya samar da fiye da ninki biyu na haɓakar samfurin?Wannan yanayin yana faruwa a cikin lokacin da ba na layi ba na amsawar PCR, wato, akwai adadi mai yawa na haɓakawa na musamman.

lankwasa narkewa
Bayan an gama haɓaka qPCR, samfurin PCR yana zafi.Yayin da zafin jiki ya tashi, samfurin ƙarawa mai ɗaci biyu a hankali yana narkewa, yana haifar da raguwar ƙarfin haske.Lokacin da aka kai wani zazzabi (Tm), yawancin samfuran za su narke.Fluorescence yana faɗuwa sosai.Kayayyakin PCR daban-daban suna da ƙimar Tm daban-daban da yanayin zafi daban-daban, ta yadda za'a iya gano takamaiman PCR.

Lanƙwan narkewa
An samo tsarin narkewa don samar da taswirar kololuwa, wanda zai iya nuna da hankali ga yanayin gutsuttsarin samfurin PCR.Tun da zafin jiki na narkewa shine ƙimar Tm na guntun DNA, ana iya tantance wasu sigogi waɗanda ke shafar ƙimar Tm na guntun DNA, kamar girman guntu, abun ciki na GC, da sauransu. Gabaɗaya magana, bisa ga ƙa'idodin ƙirar mu na farko.Tsawon samfurin da aka ƙara yana cikin kewayon 80-300bp, don haka zafin narkewa ya kamata ya kasance tsakanin 80 ° C da 90 ° C.

Fassarar maƙarƙashiyar narkewa: Idan kawai babban kololuwar ya bayyana tsakanin 80 ° C-90 ° C, yana nufin cewa PCR mai kyalli ya cika;Idan babban kololuwar ya bayyana tsakanin 80°C-90°C da kololuwar kololuwar sun bayyana kasa da 80°C, ana la’akari da dimer na farko.Kuna iya ƙoƙarin ƙara yawan zafin jiki na annealing don magance shi;idan babban kololuwar ya bayyana tsakanin 80°C-90°C, kuma kololuwar kololuwar ta sake bayyana lokacin da zafin jiki ya tashi, ana la'akari da cewa akwai gurɓataccen DNA, kuma ana buƙatar cire DNA a matakin farko na gwaji.

Tabbas, har yanzu akwai wasu yanayi mara kyau, waɗanda za a rushe ɗaya bayan ɗaya a ƙasa.
3. Nagartaccen ilimi

Don yin qPCR, dole ne in faɗi MIQE,Mafi ƙarancin Bayanidon Bugawa naƘididdigaReal-Time PCRGwaje-gwaje-mafi ƙarancin bayani don buga labarai game da ainihin-lokaci mai ƙididdige PCRgwaje-gwaje .Domin saukaka fahimtar kowa, za mu sauƙaƙa mabuɗin abun ciki.

Kuna iya bincika ainihin rubutun MIQE akan Intanet, kuma mafi mahimmancin abu shine cewa ya ƙayyadelissafin bayanan da ake buƙatar bayarwa lokacin buga labarin .

Masu dubawa za su iya yin la'akari da ingancin gwajin ta hanyar karanta waɗannan cikakkun bayanai;Masu karatu na gaba kuma za su iya amfani da wannan don maimaita ko inganta gwajin.
Ya kamata a lura cewa a cikin wannan jerin, muhimmancin kowane jeri yana da alamar E ko D bi da bi.Me ake nufi?E: mahimman bayanai (dole ne a ƙaddamar da su);D: bayanin da ake so (samar da yadda zai yiwu).

MIQE (1) — Zane na Gwaji
Yawancin ’yan iska da suka gama kare kansu bayan kammala karatunsu na digiri ba za su san yadda za su tsara gwaji da kansu ba, buɗe littattafansu, da yin abin da malamin ya ce su yi.A sakamakon haka, zanen gwajin bai kasance mai tsauri ba, kuma sashen edita na mujallar ya ce suna so su tsara wannan hoton da wancan hoton, don haka suka yi shi cikin rudani.Wannan shi ne yadda ake yin almubazzaranci!

Kusa da gida, ka'idar farko na gwaji shine ƙayyadetsananin ma'anar gwaji.Abu mafi mahimmanci shine ƙirar gwaji, kuma mafi mahimmanci game da ƙirar gwaji shine yadda za a saita samfurin da aka yi niyya, samfurin tunani (masu sarrafawa), da adadin maimaitawa, ta yadda za a iya yin la'akari da bayanan gwaji, kwatanta, da kuma gamsarwa.

Samfurin manufayana nufin samfurin da ke buƙatar mu gano ainihin kwayar halitta bayan wani magani.Misalin tunanishine samfurin ba tare da wani magani ba, wanda galibi ana kiransa nau'in daji a ilmin halitta.

Kwafi na gwajisuna da matukar muhimmanci.Gabaɗaya, adadin kwafin lallashi dole ne ya wuce uku.Wajibi ne a bambance abin da ke tattare da kwafin halittu da abin da yake kwafin fasaha.

Kwayoyin Halittu: Gwajin tabbatarwa iri ɗaya da aka yi tare da kayan daban-daban (lokaci, tsire-tsire, batches, faranti na amsawa).

Kwafin Halittu
Mu dauki maganin kashe kwari da barkono a matsayin misali.Muna so mu fesa magungunan kashe qwari a kan tsire-tsire guda uku na ABC, to, tsire-tsire uku na ABC nau'ikan halittu ne guda uku, kuma gwajin tabbaci iri ɗaya ne da aka yi da kayan daban-daban.Amma a matsayin gwaji, babu shakka ana buƙatar sarrafawa, don haka za mu iya fesa ɗaya daga cikin rassan shuka A don samar da rukunin gwaji na shuka A, ba kuma a fesa sauran rassan shuka A don kafa ƙungiyar sarrafawa ba.Yi haka don B da C.

Kwafi na Fasaha (Kwafi na Fasaha): Wani gwaji ne da aka maimaita shi don gujewa kurakurai da aiki ke haifarwa, wanda a zahiri ramin kwafi ne da aka haɗa a cikin abu ɗaya.Dukansu jiyya da sarrafawa dole ne su sami saitunan kwafi (mafi ƙanƙanta uku) na jigon da aka yi niyya da kuma jigon tunani na ciki.

Maimaituwar fasaha
Ɗauki barkono da aka yi da magungunan kashe qwari a matsayin misali kuma.Don rukunin gwaji na shuka A, mun sanya ramukan PCR guda uku na 1, 2, da 3 don asalin abin da aka yi niyya da kuma jigon tunani na ciki bi da bi, don ɗaukar matsakaicin bayan ganowa.Don kula da shuka A Ƙungiyoyin kuma ana bi da su ta hanya ɗaya.Hakazalika, yi magani iri ɗaya don tsire-tsire na B da C.Wannan maimaitawar fasaha ce.

Yana da kyau a lura da hakanAbin da ke shiga cikin kididdiga shi ne maimaitawar halittu, kuma maimaitawar fasaha ita ce a gwada ko akwai wasu abubuwan bazuwar a cikin tsarin gwaji, ta yadda za a tabbatar da sakamakon gwajin ya tabbata, wato, don guje wa kurakurai ta hanyar ɗaukar matsakaicin su kamar yadda muke yawan faɗa.

Sarrafa mara kyau-NTC da NRT
NTC (Babu-Samfurin Samfura), ana amfani da sarrafawa ba tare da samfuri ba, don tabbatar da ko kayan gwajin ya gurɓace.Gabaɗaya, ana amfani da ruwa azaman samfuri.Idan akwai amsa mai kyalli, yana nuna cewa gurɓataccen acid nucleic ya faru a cikin dakin gwaje-gwaje.

Wadannan gurbacewar yanayi sun fito ne daga: ruwa maras kyau, reagents marasa cancanta dake dauke da DNA na zamani, gurbacewar yanayi, gurbacewar kayan aikin dakin gwaje-gwaje, gurbacewar iska, da dai sauransu, suna buƙatar amfani da masu hana ruwa na RNase da masu hana RNase.Gurbacewar iska ita ce mafi wahalar samu.Ka yi tunanin dakin gwaje-gwajenka yana kama da hayaki, tare da nau'ikan acid nucleic da aka dakatar a cikin iska.

NRT (Babu Komawa Rubutu), sarrafawa ba tare da juyar da juyi ba, shine RNA mara juyi da aka rubuta a matsayin iko mara kyau, wanda shine sarrafa ragowar gDNA.

Lokacin yin bayanin kwayoyin halitta, ana gano adadin RNA ta gano adadin cDNA bayan an juyar da rubutun.Idan akwai ragowar gDNA lokacin da aka tsarkake RNA, zai haifar da kurakurai a cikin sakamakon gwaji, saboda ainihin sakamakon da aka samu shine gDNA da cDNA.A jimlar matakin, ba kawai cDNA ba, gDNA yana buƙatar cire gaba ɗaya yayin hakar RNA.

MIQE (2) — samfurin bayanai
Abin da ake kira bayanin samfurin yana nufin cewa lokacin da muka buga labarin game da qPCR, dole ne mu bayyana samfurin bayanan a sarari, wanda wani yanki ne mai mahimmanci na labarin.Hakazalika, lokacin da muke aiwatar da samfurori, dole ne mu daidaita ayyukanmu don tabbatar da ingancin samfuran.

Bayanin samfurin sakamako ne kawai, kuma ya kamata mu mai da hankali ga kayan da aka ɗauka yayin gwajin duka.

Zaɓin kayan gwaji
Samfuran jini - zaɓi sabon jini, bai wuce sa'o'i 4 ba.Samfuran tantanin halitta – zaɓi tattara sabobin sel a cikin lokacin girma mai ƙarfi.Naman dabba - Zaɓi sabo, nama mai girma da ƙarfi.Naman Shuka - Zaɓi sabo, nama matasa.

Dole ne ku lura cewa akwai wata maɓalli a cikin waɗannan ƴan jimlolin: sabo .
Don samfuran da ke sama, mafi kyawun, farashi mai tsada, kuma kit ɗin tsayayye akan kasuwa shine kayan aikin Foregene, wanda zai iya fitar da DNA da RNA cikin sauri da sauƙi.

Mini Kit na jini DNA

Salon Warewa Kit ɗin RNA

Jimlar Dabbobin Keɓewar RNA

Jumlar Shuka Kayan Keɓewar RNA

Plant Total RNA keɓewar Kit Plus

Kit ɗin keɓewar DNA na Shuka

Adana kayan gwaji
Gabaɗaya magana, ba mu bayar da shawarar adana samfuran ba, idan yanayi ya yarda.Duk da haka, akwai abokai da yawa waɗanda ba za su iya gudanar da gwaje-gwaje ba nan da nan bayan samfurin, kuma wasu ma suna buƙatar ɗaukar tankunan ruwa na nitrogen zuwa filin don yin samfur.

Don irin wannan aboki mai aiki tuƙuru, zan iya cewa ba ku fahimci abubuwan da ake amfani da su ba.Yanzu yawancin kamfanoni masu amfani da reagent suna samar da reagents waɗanda za su iya adana samfuran RNA a zafin daki, kuma zaku iya zaɓar amfani da su.Hanyar ajiya ta al'ada ita ce ajiyar ruwa ta nitrogen, ta amfani da ƙaramin tanki na nitrogen mai sauƙi wanda yake da sauƙin ɗauka.Bayan dawo da samfurin zuwa dakin gwaje-gwaje, adana shi a cikin firiji -80 ° C.

Don gwaje-gwajen da suka shafi RNA, dole ne a bi ƙa'idar kalmomi shida:low zazzabi, babu enzymes,kumasauri .

Ma'anar ƙananan zafin jiki yana da sauƙin fahimta;ba tare da enzymes ba, RNase yana ko'ina a cikin duniyar da muke rayuwa a ciki (in ba haka ba HIV ya kashe ku), don haka yadda za ku guje wa RNase lokacin yin gwaje-gwaje yana da mahimmancin mahimmanci;sauri,Babu Kung Fu a duniya wanda ba zai iya karya ba, kawai gudun ba zai iya karya ba.

Saboda haka, a wata ma'ana, guntun lokacin hakar, mafi kyawun kayan.Me yasaForegene's kit jaddada gudun, domin sun san shi da kyau.

PS: Wasu 'yan mata suna yin gwaje-gwaje a hankali, amma ba su da kyau kamar kullun kullun bayan shekaru da yawa na aiki.Suna jin cewa Allah ba ya adalci, yana gunaguni game da wasu, kuma yana neman rai.Hasali ma ba ta gane ba.Bai kare RNA da kyau ba, kuma dan wasan slam dunk yana da hankali.Lokacin da yake yin gwajin, sai ya yi tunanin cewa zai gama dalla-dalla sau uku, sau biyar da kashi biyu, amma ya yi gwajin da kyau.

Lura: A hankali, ƙarin damar mamaye RNase.Yaya za ku horar da kanku don yin sauri?Babu wata hanya, kawai ƙara yin aiki.

Don gwaje-gwaje daban-daban da samfurori daban-daban, har yanzu yana da mahimmanci don karanta ƙarin wallafe-wallafen kuma zaɓi hanyar da ta dace don sarrafawa.Don tsarin tattarawa da adanawa, MIQE yana buƙatar cewa dole ne a rubuta shi a fili a cikin takarda, don masu dubawa su sake nazarin amincin takardar, kuma yana da dacewa ga matasa masu ban mamaki su maimaita gwajin ku.

Kodayake gwaje-gwajen nazarin halittu suna da wahala, suna da tsayi.Idan ba ku yi hankali ba, kuna iya jujjuya duniya.Misali, sanya SARS cikin rikicin sinadarai, ko yin shinkafa shinkafa don ceton mutane biliyan 1.3.Hoton da ke ƙasa gwajin sinadari ne, ya kamata ku fahimci yadda kuke alfahari da bincikenku kawai ta hanyar kallon kamannin sa na dick.Manta shi, kar a yi masa baki.

MIQE (3) - hakar nucleic acid.
Hakar acid nucleic wani babban lamari ne, kuma duk gwaje-gwajen nazarin halittu suna farawa da cirewar acid nucleic.Da farko, bari mu kwafi abubuwan MIQE akan hakar acid nucleic.

Duban wannan fom, ba za ku iya zama a saman ba.Siffar akida ce.Don zama babban ɗalibi, dole ne ku tambayi dalili.Muhimmin abun ciki na wannan tebur shine: Bitsafta, mutunci, daidaito, da adadin cirewar RNA .

Kashi na farko natsari ko kayan aiki shine matakin hako acid nucleic.Idan kayi amfani da mai cire acid nucleic ta atomatik don cirewa (ci gaba, da fatan za a tuntuɓe ni don siye), kuna buƙatar nuna sunan samfurin kayan aikin.

Sunan kit da

abin da kit ɗin da aka yi amfani da shi don cikakkun bayanai na canji, abin da aka ƙara reagents na musamman ko kuma abin da aka yi na musamman ya kamata a bayyana shi a sarari domin wasu su iya maimaita gwajin ku cikin sauƙi.

Wasu mutane suna ƙara wasu reagents na musamman lokacin fitar da samfurori na musamman, suna tunanin cewa wannan makamin sirri ne kuma kada ku gaya wa wasu.Yayin da suke ɓoye shi, suna kuma rasa damar sanya labarin ku ya haskaka.Kada ku kasance da wayo, dole ne ku kasance da gaskiya fiye da tsohuwar ƙasar Zhang a cikin binciken kimiyya, idan kuna son yin wayo, labarin zai sa ku zama wawa.

dole ne a tuna da lambar samfur na kitlokacin da kuka yi odar kit ɗin kuma ku rubuta labarin.Gabaɗaya akwai lambobi biyu akan kit: Cat—lambar katalogi (lambar samfur, lambar labarin), Lot—lambar samfura (Ana amfani da shi don nuna wace tsari samfurin ya fito).

Bugu da kari, ana yawan amfani da lambar CAS lokacin yin odar reagents na biochemical, kuma zan yada shi tare.Lambar CAS ita ce lambar da Ƙungiyar Kiwon Lafiyar Jama'a ta Amirka ta ba kowane sabon sinadari.Gabaɗaya, lambobi uku ana haɗa su ta dash.Lambar CAS ta Rushui: 7732-18-5.Chemicals galibi suna da laƙabi da yawa, amma lambar CAS ta bambanta.Lokacin yin odar magani, zaku iya duba lambar CAS ta farko.

Kusa da gida, me ya sa za mu bayyana waɗannan abubuwa a fili?A gaskiya ma, shi ne kuma don duba ingancin hakar RNA.Yin amfani da kayan aiki da kayan aiki zai sa hakar RNA ya zama daidai.Ma'aunin hakar na dakunan gwaje-gwaje na yau da kullun ba su da girma, kuma ana iya samun shi tare da kits.

Cikakkun bayanai na maganin DNAse ko RNase
Muhimmin batun PCR mai kyalli shine don hana gurɓacewar DNA, kuma kada kuyi gwaji idan akwai gurɓatawa.Saboda haka, yana da mahimmanci a bayyana tsarin da kuka yi amfani da shi don sarrafa DNA, don nuna cewa an cire DNA a cikin tsarin gwaji gaba daya kuma gaba daya.wanda aka wakilta ta hanyar zane-zane.

Tsarin tsari na RNA da DNA
Gabaɗaya, hanyar cire DNA shine a bi da RNA tare da DNA bayan hakar.Duk da haka, waɗannan su ne in mun gwada da tsoffin hanyoyin.Kayan aikin hakar RNA na kasuwanci sun sami damar cire DNA yayin aikin hakar ba tare da ƙara DNA ba.Misali, jerin kayan aiki daga Foregene.

Lura: Cire DNA yayin da ake hakar RNA wani takobi ne mai kaifi biyu mai hatsarin gaske, wanda zai tsawaita lokacin aikin hakar RNA kuma yana ƙara haɗarin lalata RNA.Ainihin, ciniki ne tsakanin yawan amfanin RNA da tsarki.

Bugu da ƙari, adadin DNA ɗin da aka ƙara zuwa ginshiƙi na tushen silica yana da ƙananan ƙananan, kuma dole ne a yi amfani da DNase mai inganci don cimma sakamako.DNase mara inganci ba za a iya narkar da shi cikin sauri da gaba ɗaya ba.Wannan gwaji ne na matakin fasaha na ɗan kasuwa.Tabbas, akwai wasu 'yan kasuwa masu ban mamaki waɗanda suka yi alfahari cewa ana iya cire DNA ba tare da DNAse ba.Ana iya cewa duk wanda ya yi taƙama cewa DNA za a iya cire gaba ɗaya ba tare da DNAse ba, baƙar fata ce.DNA wani tsayayyen tsari ne mai tsayi biyu, kuma ba za a iya shafe shi ta hanyar magana da dariya kawai ba.

Ƙimar gurɓatawa
Hanyar tantancewa: ganowar electrophoresis, 1% agarose, 6V/cm, 15min, 1-3 ul

Nucleic acid ƙididdigar ƙididdiga
yawanci ana auna ta ta amfani da spectrophotometer UV.Bari in fara yada ma'anar darajoji uku na OD260, OD280, da OD230.
OD260nm: Ita ce tsayin daka na sha mafi girman kololuwar acid nucleic, kuma mafi kyawun ƙimar ƙima daga 0.1 zuwa 1.0.Idan ba haka ba, tsoma ko maida hankali samfurin don kawo shi cikin kewayo.
· OD280nm: shine tsayin raƙuman sha na mafi girman kololuwar furotin da abubuwan phenolic.
OD230nm: shine tsayin raƙuman sha na mafi girman kololuwar ƙwayar carbohydrates.

Na gaba, bari muyi magana game da rawar kowane mai nuna alama.Don A260, ana iya amfani dashi don auna yawan amfanin ƙasa na nucleic acid.Lokacin OD260=1, dsDNA=50μg/ml, ssDNA=37μg/ml, RNA=40μg/ml.

Don tsabta, muna buƙatar duba ƙimar da muke yawan gani: OD260/280 da OD260/230.
DNA mai tsafta: OD260/280 kusan daidai yake da 1.8.Lokacin da ya fi 1.9, yana nuna cewa akwai gurɓataccen RNA, kuma idan bai kai 1.6 ba, yana nuna cewa akwai gurɓataccen furotin da phenol.
· Tsaftace RNA: 1.7
OD260/230: Ko DNA ne ko RNA, ƙimar ma'anar ita ce 2.5.Lokacin da bai wuce 2.0 ba, yana nuna cewa akwai gurɓataccen sukari, gishiri da kwayoyin halitta.

RNA mutunci

Yana da matukar mahimmanci don auna amincin RNA.Gabaɗaya, ya zama dole a yi gwajin gel denaturation na RNA don bincika ko haske tsakanin 28S da 18S RNA dangantaka ce mai ninki biyu.Lokacin da rukuni na uku na 5S ya bayyana, yana nufin cewa RNA ta fara raguwa, sai dai invertebrates.

Bayanai don kimanta ingancin RNA: Bugu da ƙari ga gwaje-gwajen da ke sama, akwai kuma wasu ƙarin gwaje-gwajen kayan aiki na ci gaba dangane da amincin RNA, kamar gwajin amincin RQI na tsarin lantarki na atomatik na Experion, wanda zai iya gano ko RNA ya ƙasƙanta da ganuwa.

A cikin binciken kimiyya, PCR mai ƙyalƙyali shine kwatancen tsakanin jigon da aka yi niyya da kuma jigon tunani na ciki.Saboda haka, a cikin tsarin adana samfurin RNA, cirewar RNA, da dai sauransu, burin farko shine tabbatar da amincin RNA.

Yadda mutuncin RNA ke shafar ma'auni tsakanin jigon manufa da kuma jigon tunani na ciki ana iya fahimta cikin sauƙi daga wannan adadi a ƙasa.Lalacewa zai haifar da rashin cikar kwayoyin halitta, ko dai rashin cikar kwayoyin halittar da ke cikin gida ko kuma rashin cikar kwayar halittar da aka yi niyya, zai yi tasiri sosai kan bayanan.

Zane-zane na tsararrun kwayoyin halittar da aka yi niyya da kuma jigon tunani, dole ne kada ya zama gaskiya

Gwajin hanawa (ko ƙimar CT an kashe shi a ƙarƙashin babban taro ko ƙaranci ko wasu yanayi)

Daukar wannan adadi a matsayin misali, ma'aunin Ct na lankwasa biyar su ne kamar haka.Rarraba dabi'un CT tsakanin masu lankwasa ba daidai ba ne, kuma ana jinkirin ƙimar Ct a ƙarƙashin babban ƙima da ƙarancin ƙima, wanda shine yanayin hana PCR.

Mahimmin batu: A cikin aikin hakar RNA, muna buƙatar yin watsi da kuskuren fahimta kuma mu kafa daidai.

Ra'ayin da ba daidai ba shine: cirewar RNA yana bin amfanin gona ne kawai, yana tunanin cewa girman adadin RNA da aka samu, mafi kyau.A gaskiya ma, idan muka yi ƙididdigewa, idan adadin kwayoyin halitta ba su da yawa sosai, ba ma buƙatar RNA da yawa.Adadin RNA da kuke cirewa ya fi isa .

Madaidaicin ra'ayi shine:Ya kamata hakar RNA ya bi tsabta, mutunci da daidaito.Tsafta na iya tabbatar da cewa ba a hana rubutun baya ba kuma DNA ba za ta shafi bayanan ba.Mutunci yana tabbatar da ma'auni na jerin manufa da nassoshi na ciki.Daidaituwa yana tabbatar da kwanciyar hankali samfurin lodi.

MIQE (4) - juyar da rubutu
Rashin fahimta: neman mafi girma samfurin girma.
Ma'anar daidaiNeman daidaito (kwanciyar hankali), ba tare da la'akari da adadin RNA da aka ɗora ba, ingancin juzu'i ya kasance mai daidaituwa, tabbatar da cewa bambance-bambance a cikin cDNA na iya nuna bambance-bambance a cikin mRNA da gaske.
Muna bayyana wannan tsari tare da zane mai tsari:

Zane-zane na ingantaccen juzu'i, ba gaskiya bane
Da farko, muna buƙatar fahimtar bambanci tsakanin tsarin rubutun baya da tsarin PCR.PCR yana jurewa da dumama da tafiyar matakai, kuma guntun da aka yi niyya yana girma sosai;yayin da juzu'i ba shi da wannan tsari, zamu iya tunanin cewa juyar da rubutun shine ainihin ɗaya-zuwa ɗaya Yayin aiwatar da maimaitawa, kamar guda ɗaya na RNA.

kamar yadda ake iya samun adadin bayanai na cDNA, ya kamata a gane yanzu, domin an juyar da gutsuttsura manya da ƙanana, kuma ba shi yiwuwa a mai da hankali ga guntu ɗaya.Kuma saboda adadin RNA yana da ƙanƙanta, adadin cDNA da aka samu shima kaɗan ne, ba kamar PCR ba, wanda ke da tasirin haɓakawa, don haka ba shi yiwuwa a gano shi.

Sakamakon electrophoresis cDNA
Abu na biyu, a zahiri, ana yin juzu'i ɗaya-zuwa ɗaya, amma babu juyar da rubutun daga kowane kamfani da zai iya cimma wannan tasirin.Ainihin, ingancin mafi yawan jujjuya bayanan baya yawo tsakanin 30-50%.Idan haka ne, za mu gwammace mu sami ingantaccen ingantaccen rubutun juzu'i, wanda shine abin da muke son gani a cikin adadi: 3 RNAs suna samun cDNAs 2, RNAs 6 suna samun cDNAs 4, don haka komai yawan samfurin da aka ɗora, ingancin rubutun baya yana da inganci.Ba ma so mu ga halin da ake ciki inda ingancin rubutun baya ba shi da tabbas kuma an hana babban taro.

Don haka, ta yaya za a tabbatar da ko ingancin rubutun baya ya tabbata?Hanyar abu ne mai sauqi qwarai, kawai kuna buƙatar yin gwajin kwatance: ɗaya shine a juyar da rubutawa zuwa cDNA bayan dilution na RNA sau biyu, ɗayan kuma shine yin dilution biyu bayan an juyar da shi zuwa cDNA, sannan ku yi qPCR don ganin gangaren da aka samu Shin daidai ne.A matsayin babban ɗalibi, yakamata ku fahimce shi cikin daƙiƙa.Kamar yadda aka nuna a kasa:

Dilution na RNA da cDNA don gwada ko ingancin juzu'i ya tabbata
Juya rubutun da kit
Ta yaya cikakken PCR mai kyalli zai iya samun kyakkyawan juzu'i da kit.Reverse transcriptase an raba kusan kashi biyu bisa ga tushen, AMV koM-MLV, kuma aikin su daidai yake da wanda aka nuna a tebur.

RNase H aiki
RNase H shine Ribonuclease H, sunan Sinanci shine ribonuclease H, wanda shine endoribonuclease wanda zai iya yin hydrolyze RNA musamman a cikin sarkar DNA-RNA.RNase H ba zai iya yin amfani da haɗin phosphodiester a cikin DNA ko RNA mai ɗauri ɗaya ko biyu ba, wato, ba zai iya narkar da DNA ko RNA mai ɗaci ɗaya ko biyu ba.Yawanci ana amfani da shi a cikin haɗin igiyar cDNA na biyu.

Wani bakon abu ne.Mun ce reverse transcriptase yana da aikin RNase H, ba wai cewa reverse transcriptase ya ƙunshi RNase H ba, kuma maiyuwa ba zai yiwu a raba RNase H da reverse transcriptase ba, watakila saboda daidaitawar wasu ƙungiyoyi a cikin juzu'i.

Saboda haka, ba tare da la'akari da ingantaccen juzu'i na AMV ba, ayyukan RNase H yana rage yawan amfanin cDNA.Tabbas, masana'antun reagent suna haɓaka samfuran su koyaushe don kawar da ayyukan RNase H a cikin juzu'i kamar yadda zai yiwu don ƙara yawan amfanin cDNA.
zafin jiki mai raɗaɗi

Tsarin RNA na biyu a yanayin zafi daban-daban
Dubi hoton da ke sama don tsarin na biyu na RNA a yanayin zafi daban-daban, kuma yi amfani da kayan aikin kan layi na mFold don tantance tsarin na biyu na guntun manufa ƙarƙashin takamaiman yanayin zafin jiki da gishiri.A 55°C, tsarin na biyu na RNA har yanzu yana da sarƙaƙiya sosai, jujjuya bayanan baya iya aiki, kuma tsarin na biyu ba zai iya warware gaba ɗaya ba har sai 65°C, yayin da mafi kyawun zafin jiki na AMV da M-MLV yayi ƙasa da wannan zafin.
me za ayi?Tsarin na biyu shine haɗin haɗin samfurin da kansa, wanda ke haifar da gasa mai ƙarfi tsakanin madaidaicin rubutu da jujjuya rubutu da samfuri, yana haifar da jerin matsaloli kamar ƙarancin E da ƙarancin maimaitawa.

me za ayi?Sai kawai ƙara yawan zafin jiki mai raɗaɗi gwargwadon yiwuwa .

Yawancin masana'antun reagent suna haɓaka jujjuya bayanan su ta hanyar injiniyan kwayoyin halitta.Wasu suna ƙara yawan zafin jiki, kamar Jifan da Aidelai, wasu kuma suna cire rukunin aiki na enzyme na RNase H don inganta alaƙar da ke tsakanin enzyme da samfurin RNA.Babban kusanci na iya yin gasa gasa fitar da tsarin na biyu da karantawa cikin sauƙi, sannan kuma yana haɓaka ingantaccen juzu'i.
Mahimmin mahimmin abu: Rubutun juzu'i ya fi mahimmanci don biyan daidaiton ingancin juzu'in juzu'i (enzymes dole ne ba kawai su kasance masu inganci ba amma har ma sun tsaya tsayin daka), maimakon adadin samfurin da aka ɗora, idan ba babban girman girman girman PCR ba, ba zai yiwu ba kwata-kwata.Yawancin cDNAs.
Masana'antun daban-daban sun kuma yi wasu yunƙuri don neman daidaito.Misali, yawancin kamfanoni yanzu sun tattara rubutun juzu'i a matsayin daidaitaccen kayan siyarwa, wanda zaɓi ne mai kyau.
Misali, Foregene's RT Easy Series kits:

RT Easy I (Master premix na farko da igiyar cDNA kira kit)

MIQE (5) - bayanin jinsin manufa

Adadin da ke sama yayi bayani
1. Ko wannan kwayar halitta tana da tasiri don maimaita gwaje-gwaje ana iya tabbatar da ita gabaɗaya ta maimaita gwaje-gwaje.
2. Gene ID, kun sani.
3. Tsawon Halittar Halittar Halittar Halittar Halittu, jimillar adadin da aka yi niyya babu shakka babu matsala.Lokacin zayyana firamare, tabbatar da cewa tsawon amplicon yana tsakanin 80-200bp don tabbatar da ingantaccen haɓakawa.
4. Bayanin kwatanta fashewar jerin abubuwa, ana buƙatar kwatanta kwayar halittar da aka yi niyya a banki ta gene don hana haɓakawa da ba takamaiman ba.
5. Kasancewar pseudogenes.Pseudogene jerin DNA ne mai kama da na al'ada amma ya rasa aikinsa na yau da kullun.Yana sau da yawa a cikin Multi-Gene iyali na eukaryotes.Yawancin lokaci ana wakilta ta ψ.Kwafin DNA ne wanda ba ya aiki a cikin kwayoyin halitta wanda yayi kama da jerin kwayoyin halittar coding., gabaɗaya ba a rubuta su ba, kuma ba su da ma'ana ta zahiri.
6. Matsayin farko dangane da exons da introns.A cikin shekarun farko, lokacin da muka magance matsalar gurɓataccen DNA, sau da yawa muna mai da hankali ga matsayi na firamare, exons, da introns, kuma gabaɗaya muna yin la'akari da zayyana abubuwan ƙira a cikin introns don guje wa haɓaka DNA.Da fatan za a duba hoton da ke ƙasa: baƙar fata yana wakiltar introns, shuɗi daban-daban suna wakiltar exons, ruwan hoda yana wakiltar al'amuran gama-gari, ja mai haske yana wakiltar firamare masu faɗin shiga.

Tsarin tsari, ba gaskiya bane
Menene cikakken shirin wannan alama, amma a zahiri, a mafi yawan lokuta, trans-intron primers ba su da sihiri kamar yadda aka yi zato, kuma za su haifar da haɓakawa na musamman.Don haka hanya mafi kyau don hana gurɓacewar DNA ita ce cire DNA gaba ɗaya.
7. Hasashen daidaituwa.Amfani da wannan misalin kuma, yi amfani da kayan aikin kan layi na mFold don tantance tsarin na biyu na guntun manufa a takamaiman zafin jiki da maida gishiri.

Tsarin RNA na biyu a yanayin zafi daban-daban
Tsarin na biyu shine haɗin haɗin samfurin da kansa, wanda zai haifar da gasa mai ƙarfi tsakanin na'ura mai ƙima da haɗawa da samfuri, kuma damar daurin firam ɗin ba ya da ƙasa, yana haifar da jerin matsaloli kamar ƙarancin E da rashin maimaitawa.Ta hanyar tsinkayar software, idan babu matsalar tsari na biyu, hakan zai yi kyau.Idan akwai, labarinmu na gaba zai tattauna musamman yadda za a magance wannan matsalar.

MIQE (6)-qPCR Oligonucleotides

Don PCR mai ƙyalƙyali, abu na farko da kuke kokawa da shi kowace rana shine cirewar RNA, kuma abu na biyu na iya zama ƙirar farko.
Da farko, har yanzu muna bincika ƙa'idodi game da ƙira na farko bisa ga jerin abubuwan MIQE.Abu ne mai sauƙi wanda masu ɓarna za su iya yin dariya, kuma za mu iya gama shi a cikin jumla ɗaya: gano jerin da matsayi na binciken farko da hanyar gyarawa.Don hanyar tsarkakewa na farko, ƙaddamar da ƙaddamarwa yana da arha a halin yanzu, qPCR ya cancanci PAGE kuma sama da hanyoyin tsarkakewa, kuma bayanin kayan aikin haɗin gwiwar ba shi da mahimmanci.Mutane da yawa sun kasance suna yin abubuwan farko na shekaru da yawa kuma basu san cewa mai haɗawa shine ABI3900 ba.
Game da ka'idodin ƙirar ƙira, ba dole ba ne ka haddace su ta hanyar rote, saboda yawancin software na ƙira ko kayan aikin kan layi na iya magance waɗannan matsalolin (shawarar kayan aikin kan layi na 3.ut.ee/), da kuma 99.999% na ƙirar ƙira ba a yi da hannu Dubi, marubucin wani lokacin yana tsara ɗaruruwan abubuwan ƙira a rana, idan kun karanta ɗaya bayan ɗaya, zai zama giciye.
Kawai duba abubuwan da suka biyo baya bayan an tsara abubuwan farko:
1. Ƙirar ƙirar ƙira kusa da ƙarshen 3': A cikin yanayin yin amfani da oligo dT masu mahimmanci don haɗin haɗin farko na cDNA, la'akari da juzu'i na juzu'i da amincin RNA, abubuwan da aka tsara suna buƙatar a tsara su kusa da ƙarshen 3' don inganta haɓaka haɓakawa.Yi amfani da hoto don bayyana kamar haka (babu yadda za a fahimci wannan):

Me ya sa za a ƙera firamare kusa da ƙarshen 3′, ba dole ba ne gaskiya
2. Darajar TM: Darajar Tm yana a 55-65 ° C (saboda aikin exonuclease shine mafi girma a 60 ° C), kuma abun ciki na GC yana a 40% -60%.
3. BLAST: Don guje wa haɓakar ƙwayoyin halitta marasa takamaiman, dole ne a yi amfani da fashewa don ƙarin tabbaci.

MIQE(7)-qPCR tsari

1. qPCR kit
Dangane da buƙatun MIQE, dole ne mu bayyana a sarari cikakken yanayin amsawa a cikin labarin, gami da daidaitawar tsarin amsawar PCR, menene kit ɗin da aka yi amfani da shi, wanda shine masana'anta, girman tsarin amsawa, ko ana amfani da hanyar rini ko hanyar bincike, saitunan shirin PCR.Tsohuwar direbobi tabbas za su gano cewa muddin aka zaɓi kayan aikin, ainihin bayanan da ke sama an ƙaddara su.
A halin yanzu, ƙira da samar da na'urorin PCR masu kyalli da yawa fasaha ce da ta balaga.Muddin ba za ku zaɓi masana'anta marasa kyau ba, yiwuwar matsalolin ba su da yawa, amma har yanzu muna so mu raba tare da ku 'yan maki:
Hot-farawa Taq enzyme:Mafi mahimmancin ɓangaren PCR shine enzyme Taq mai zafi.Enzymes masu zafi-farawa akan kasuwa gabaɗaya sun kasu kashi biyu, ɗayan shine ingantaccen ingantaccen enzyme mai farawa mai zafi (zaku iya tunanin shi azaman shigar da paraffin), ɗayan kuma shine Enzyme mai zafi-farawa don gyaran antibody (antigen-antibody binding).Gyaran sinadarai hanya ce ta farko ta enzymes masu zafi.Lokacin da wani zafin jiki ya kai, enzyme zai saki aikinsa.Enzyme farawa mai zafi da aka gyara-antibody yana amfani da hanyoyin nazarin halittu don toshe ayyukan enzyme.Lokacin da wani zafin jiki ya kai, za a cire antibody kuma a daina kunna shi azaman furotin, kuma aikin enzyme zai shiga cikin wasa.

Duk da haka, menene amfanin wannan?Wannan shine lamarin, aikin sakin enzymes wanda aka gyara ya fi sauri fiye da na enzymes da aka gyara ta hanyar sinadarai, don haka dangane da hankali, enzymes ɗin da aka gyara na antibody yana da ɗan fa'ida, ta yadda a zahiri babu enzymes ɗin da aka gyara a cikin kayan a kasuwa.Idan akwai, to fasahar masana'anta har yanzu tana makale a zamanin karni.
Magnesium ion maida hankali:Matsakaicin ion magnesium yana da mahimmanci a cikin halayen PCR.Mahimmancin maida hankali na ion magnesium na iya haɓaka sakin ayyukan enzyme Taq.Idan maida hankali ya yi ƙasa sosai, aikin enzyme zai ragu sosai;idan maida hankali ya yi yawa, za a inganta haɓakar haɓakar da ba ta da takamaiman enzyme-catalyzed.Matsakaicin ions na magnesium kuma zai shafi ɓarkewar abubuwan haɓakawa, yanayin narkewar samfuri da samfuran PCR, ta haka yana shafar yawan ɓangarorin da aka haɓaka.Matsakaicin ions na magnesium gabaɗaya ana sarrafa shi a 25mM.Tabbas, don kit mai kyau, ƙaddamar da ions na magnesium dole ne a sarrafa shi da kyau.Wasu 'yan kasuwa suna ƙara wakili na chelating na magnesium ion zuwa reagent, wanda zai iya cimma tasirin daidaitawa ta atomatik na ƙwayar magnesium ion.
Matsakaicin rini na Fluorescent:Rini na Fluorescent, wanda shine SYBR Green da muke amfani da shi, galibi yana haifar da haske ta hanyar ɗaure ga ƙaramin tsagi na DNA mai ɗauri biyu, saboda ɗaurin rini zuwa DNA mai ɗauri biyu ba takamaiman ba ne, ma'ana Muddin DNA ɗin mai ɗaure biyu ta haɗu da shi, ƙirar fluorescence na iya faruwa a cikin siginar na'ura da tsarin DNA.
PS: Saboda kaddarorin sa na haske, samfuran kan kasuwa gabaɗaya ana tattara su cikin bututun santsi mai launin ruwan kasa (kamar yadda aka nuna a hoton da ke ƙasa).Duk da haka, wannan zai fuskanci matsala.Yana da wuya a ga ko an sha ruwa lokacin yin samfur.Dangane da wannan, Qingke ita ce ta fi dacewa da masu amfani (kamar yadda aka nuna a hoton da ke ƙasa), kuma an haɗa bututun da ke bayyana a cikin jakar kwano mara kyau.Sa'an nan kuma sanya shi a cikin jakar kwano, la'akari da saukakawa na guje wa haske da samfurin.Dole ne ku zaɓi lambar samfur daidai.TSE204 rayuwa ce mai matukar tsada, wanda ke sa ni son shuka ciyawa.

Matsakaicin rini mai kyalli shima yana da mahimmanci.Idan maida hankali ya yi ƙasa da ƙasa, ƙirar haɓakawa ba za ta haura a mataki na gaba ba kuma ba cikakke ba;idan maida hankali ya yi yawa, zai haifar da tsangwama amo.Tun da PCR mai kyalli ya dogara da ƙimar CT, idan ba a daidaita yawan rini mai kyalli da kyau ba, ƙaramin ma'ana ya fi babban ma'ana.Tabbas, ƙaddamar da launi mai dacewa shine mafi kyau.

ROX: Ana amfani da rini na ROX don gyara kurakuran siginar walƙiya mai kyau da kyau.Wasu masana'antun kayan aiki suna buƙatar daidaitawa, yayin da wasu ba sa.Misali, amfani da Thermo Fisher Scientific's Real Time PCR amplification kayan aiki yawanci yana buƙatar daidaitawa, gami da 7300, 7500, 7500Fast, StepOnePlus, da sauransu. Umurnin kit na gabaɗaya zai bayyana shi.
Foregene's qPCR Mix shima yana ƙunshe da rini na ROX, wanda ya dace don amfani a samfura daban-daban.

Real Time PCR Kit-Taqman

Jiyya na haɗin gwiwa mai rauni: Maganin raunin hydrogen bond abu ne mai ingantacciyar fasaha.Babu wani abu da ya karanta littattafan littattafai da yawa, amma babu ɗayansu da ya ambaci wannan batu.A gaskiya ma, yana da mahimmanci.Haɗin tushe ya dogara ne akan ƙarfin haɗin gwiwar hydrogen.Ƙarfin haɗin gwiwar hydrogen shine haɓakawa na al'ada, kuma raunin hydrogen bond yana haifar da haɓakawa maras takamaiman.Idan ba za a iya kawar da haɗin gwiwar hydrogen mai rauni da kyau ba, ba za a iya kauce wa haɓakawa na musamman ba.A cikin iyakokin marubucin, ƙananan kamfanoni ne kawai suka lura da wannan matsala.Lokacin da ka sayi kayan, za ka iya komawa zuwa ko ka yi la'akari da mafita a wannan batun don kayan da kake son zaɓa.

Ƙarar martani: An fi amfani da tsarin 20-50ul, kuma ƙananan ƙididdiga na iya haifar da kurakurai.Gabaɗaya magana, umarnin kit ɗin zai ba da shawarar amfani da juzu'in amsawar PCR.Kada ku kasance masu wayo kuma kuyi amfani da ƙaramin juzu'i don adana farashi.makasudin.An gwada ƙarar da 'yan kasuwa suka ba da shawarar a zahiri, kuma yana iya yiwuwa ba za su iya magance matsalar kurakurai da ƙananan kundila suka haifar ba.
2. Mai ƙira da lambar labarin na farantin bututu
Kowa ya san ka'idar PCR mai kyalli.Ana gudanar da tarin fluorescence galibi ta hanyar bututun PCR.Lokacin zabar kayan amfani da PCR, kula da maki biyu: watsa haske mai kyau da dacewa da kayan aiki.Gabaɗaya magana, allunan da bututun samfuran samfuran al'ada suna da kyau, amma dole ne ku zaɓi a hankali dangane da daidaitawa, in ba haka ba ba za ku iya amfani da kayan aikin ba.

4. Babban ilimi

MIQE (8)-qPCR ingantacce
Wannan shine babban fifiko na qPCR!Don haka da yawa jarumai sun fada cikin rairayi a nan.Tabbas, yana yiwuwa kuma kuna da sa'a kuma kwayoyin halittar da kuka yi nazari suna da sauƙi, don haka kuna shawagi a cikin kogon kankara tare da iska.Bayanan tabbatarwa na qPCR an yi niyya ne don gwada amincin bayanan.Muna jera mahimman bayanan tabbatarwa kamar haka:

1.Specificity gwajin
Ana gwada ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun kwayoyin halitta ta hanyar duba ko hoton electrophoresis band guda ɗaya ne;tabbatar da jerin abubuwa;lanƙwan narkewa don ganin ko taswirar kololuwa ɗaya ce;Tabbatar da narkewar enzyme da sauran hanyoyin.
A nan, mun mayar da hankali kan tya bincikar haɓakar da ba ta dace ba ta amfani da hanyar narkewa.Gabaɗaya magana, lokacin da muke ƙirƙira abubuwan ƙira, ana buƙatar girman guntun samfurin ya kasance cikin kewayon 80-200bp, wanda ke sanya zafin narkewar samfurin PCR 80-85 °C kewayon.Don haka, idan akwai kololuwa daban-daban, dole ne a sami wasu samfuran ƙarawa waɗanda ba takamaiman ba;idan kololuwar ya bayyana a kasa 80 ° C, ana la'akari da shi azaman dimer na farko;idan kololuwar ya bayyana sama da 85°C, gabaɗaya ana ɗaukarsa a matsayin gurɓatawar DNA ko ƙarin ƙaƙƙarfan ƙaƙƙarfan ƙaƙƙarfan guntu.
Lura: Wani lokaci ana samun kololuwa ɗaya kawai a 80 ° C.A wannan lokacin, dole ne a kiyaye wannan ra'ayi.Yana yiwuwa sakamakon ƙarawa duk na'urar dimers ne.

Narkewar al'ada (kololuwa guda ɗaya ba tare da ƙaƙƙarfan ƙarawa ba)

Matsalolin narkewar lanƙwasa (ba takamaiman ƙaƙƙarfan ƙaƙƙarfan kololuwa ba)
【Binciken shari'a】

Akwai babban kololuwa, amma dimer na farko yana da mahimmanci
Ƙunƙarar narkewa ɗaya-kololuwa a cikin adadi da ke ƙasa zai iya yaudarar idanunku cikin sauƙi, kuna tunanin cewa gwaji ne cikakke, amma sakamakon gaba ɗaya kuskure ne.A wannan lokacin, dole ne mu kalli yanayin zafi na narkewa.Matsakaicin zafin jiki yana ƙasa da 80 ° C, wanda shine gaba ɗaya-dimer.

Babu guntun manufa, duk dimers na farko
Anan, yayana ba zai iya tsayawa ba.Hoton da ke ƙasa hoto ne da aka ɗauka tare da wayar hannu da wani ɗan iska ya aiko min.Reagents da ya yi amfani da su duk samfuran da aka saba amfani da su a masana'antar.Ya canza daga alamar T-prefix ɗaya zuwa wata alamar T-prefix.Ina tsammanin kun riga kun yi hasashe.Ƙwararriyar ta yi mini kuka: “Mai amfani da reagent da aka yi amfani da shi a hoton farko ya yi kyau sosai, kuma kololuwar ba ta da aure.Daga baya, bayan amfani da reagent da kuka ba da shawarar, ya zama kamar hoto na biyu, tare da gauraye kololuwa.Kun sanya ni cikin bacin rai."
Rarrabe jadawali biyu.A kallon farko, ɗayan yana da kololuwa ɗaya, ɗayan kuma yana da kololu biyu.Maganar banza, kololuwa daya ba shakka yana da kyau.Shin gaskiya ne?
Mafi muni fiye da Dou E, idan na sanya hotuna biyu a hoton da ke ƙasa, za ku gane nan da nan.A gaskiya ma, irin wannan hoton yana gurgunta mu cikin sauƙi.Bayan bincike mai zurfi, mun gano cewa: kololuwar adadi na farko yana a 75 ° C, wanda shine cikakkiyar dimer;kololuwar adadi na biyu yana bayyana a 75°C da 82°C, aƙalla akwai samfurin ya bayyana.

Hotunan martani daga ɗalibai
Don haka matsala ta asali ba shine matsalar reagents ba, amma matsalar ƙirar ƙira.A lokaci guda kuma, yana tabbatar da cewa wasu manyan samfuran ba su da ingancin ƙarfe, kuma yana tabbatar da abin da ɗan'uwana ya faɗa a baya: Ba alamar reagent ba ce ke goyan bayan labarin ku.Labarin ku ne ya samar da alamar reagents.Ka yi tunanin, idan scumbag bai canza reagents ba, za a aika da bayanan da ba daidai ba zuwa jarida, kuma abin da zai faru zai zama bala'i.
2. Ct ƙimar kulawa mara kyau
Kar a yi bayani, idan ikon da ba komai yana da darajar Ct, ba gurbatawa ba ne?Duk da haka, har yanzu kuna buƙatar fahimtar wane iko mara tushe yana da ƙimar Ct.Idan NTC ne, yana nufin cewa akwai DNA na ƙasashen waje kamar gurɓataccen reagent.Idan NRT ne, yana nufin cewa RNA da aka cire yana da gurɓataccen DNA.
3. Daidaitaccen lankwasa
Ciki har da gangara da dabarar lissafi, ana iya ƙididdige ingancin PCR ta hanyar dabara.Cikakken gwaji yana buƙatar gangaren madaidaicin lanƙwasa don kusanci 3.32, da R² don kusanci 0.9999.
4. Mizani mai ƙarfi mai ƙarfi
Matsakaicin kewayon amsa shine madaidaiciya.Dangane da samfurin da aka yi amfani da shi don samar da madaidaicin madaidaicin, kewayon mai ƙarfi yakamata ya haɗa da aƙalla gradients na hankali 5, kuma a kula da canjin ƙimar Ct a babban matakin maida hankali da ƙarancin hankali.
5. Gano daidaito
Canje-canje a sakamakon qPCR, wato, rashin maimaitawa, wato, rashin daidaituwa, ana haifar da shi ta hanyar abubuwa da yawa, ciki har da zafin jiki, maida hankali, da aiki.Madaidaicin qPCR gabaɗaya ya zama ƙasa da abin sarrafawa yayin da lambar kwafin ta ragu.Mahimmanci tsakanin-bambancin gwaji, wannan bambancin fasaha ya kamata ya bambanta da bambancin ilimin halitta, kuma kwafin halittu na iya magance bambance-bambancen ƙididdiga kai tsaye a sakamakon qPCR tsakanin ƙungiyoyi ko jiyya.Musamman don ƙididdigar bincike, mafi kyawun madaidaicin tsaka-tsakin tsaka-tsakin (maimaitawa) a cikin rukunin yanar gizo da masu aiki dole ne a ba da rahoton.
6. Gano iya aiki da LOD (a cikin multiplex qPCR)
LOD shine mafi ƙarancin taro na 95% na samfuran tabbatacce da aka gano.Ma'ana, maida hankali na LOD da ke ƙunshe a cikin jerin abubuwan da aka yi niyya na kwafin kwayoyin halitta bai kamata ya wuce 5% na halayen da suka gaza ba.Lokacin yin nazari na qPCR multiplex, musamman don gano lokaci guda na sauye-sauyen ma'ana ko polymorphisms, multiplex qPCR yana buƙatar samar da shaida cewa daidaiton ɓangarorin da aka yi niyya da yawa ba a daidaita su ba a cikin bututu guda ɗaya, ganowa da yawa da kuma ganowar bututu guda ɗaya Ingantaccen kuma LOD ya kamata ya zama iri ɗaya.Musamman ma lokacin da aka haɓaka manyan abubuwan da aka yi niyya da kuma ƙananan ƙwayoyin cuta a lokaci guda, dole ne a kula da wannan matsala.
Matsaloli da mafitaGabaɗaya magana, matsalolin da ake fuskanta sau da yawa a cikin qPCR debugging suna mai da hankali kan abubuwa masu zuwa:
· haɓakawa maras takamaiman
Zaɓan mai wahala na maida hankali na farko da matsala tare da na'urar dimers
Zazzaɓin zafi ba daidai ba ne
Tsari na biyu yana rinjayar ingancin haɓakawa
haɓakawa maras takamaiman
ƙarawa ba takamaiman bayana faruwa , ana la'akari da shi ko ƙirar ƙirar ƙira ba ta dace ba, amma idan ba ku da sauri don canza masu farawa, zaku iya gwada waɗannan hanyoyin da farko (ka'idar kuma tana haɗe):
Ƙara yawan zafin jiki mai ban sha'awa - yi ƙoƙarin yin raunin hydrogen bond ba zai iya kiyayewa ba;
· Gajeren ɓacin rai da lokacin haɓakawa - rage damar raunin haɗin gwiwar hydrogen;
Rage maida hankali na farko - rage damar dauri na abubuwan da ba a iya amfani da su ba da kuma yankuna marasa manufa;
Ƙarfin haɓakawa
Akan lamiri don ba takamaiman amplification - low amplifiation, da kuma matakan yin amfani da ƙarancin ingancin amplifiation ne kawai:
· Tsawaita lokacin cirewa da haɓakawa;
Canji zuwa PCR mataki uku kuma rage yawan zafin jiki;
·Ƙara ƙaddamar da ƙaddamarwa;
Ps: Yawancin ɗaliban da suka kammala karatun digiri waɗanda aka haife su a cikin 90s ba sa son yin nazarin yadda za a lalata gwaje-gwajen, kuma suna fatan cewa kit ɗin zai iya magance matsalar gaba ɗaya (idan kuna son zuwa kamfanin reagent don yin bincike da haɓakawa bayan kammala karatun), a zahiri, masana'antun reagent kuma suna tunanin wannan hanyar, Ina fata yana da wawa Ana iya amfani dashi lokacin da kuka samu, don haka reagent na haɓakar haɓakawa, gami da haɓakar haɓakar haɓakar haɓakar haɓakar haɓakawa da yawa, gami da haɓakawa da haɓakawa da haɓakawa da haɓakawa da haɓakawa da yawa. Abubuwan sha na H-bond.Don magance matsalar cikin sauƙi, wawaye har yanzu suna karanta gabatarwar kamfanin reagent don ganin ko akwai wani abu da ke ɗaukar raƙuman haɗin gwiwar hydrogen.
Zaɓin mai wahala na maida hankali na farko da matsala tare da dimers-primer
Hanya 1Gabaɗaya magana, umarnin kit na qPCR sun ba da shawarar tsarin da shawarar ƙima.
Hanyar 2: Gyara kurakurai ta hanyar saita gradient na farko.Hoton da ke ƙasa an sace shi daga kamfani don kwatanta.Hoton da ke ƙasa yana nuna sakamakon ƙididdiga masu haske da aka yi tare da matakan maida hankali na asali guda uku (100nM, 250nM, 500nM) da gradients na samfuri huɗu (0.1ng, 1ng, 10ng, 100ng).An tsara ƙimar Ct na sakamakon gwajin kamar haka:

Zaɓin Ƙarfafa Tattaunawa na Farko Haɗa kowane taro na farko cikin layi kamar haka:

Zaɓin ƙaddamar da ƙaddamarwa a bayyane yake, alaƙar layi na haɗin kai na 100nM da 250nM ya fi kyau, kuma dangantakar da ke da alaƙa na ƙaddamarwa na 500nM ba shi da kyau.A cikin 100nM da 250nM, ƙimar Ct na 250nM ba ta da ɗanɗano kaɗan, don haka mafi kyawun maida hankali na farko shine 250nM.Gabaɗaya ana iya ganin firam-dimer mai tsanani a cikin lanƙwan narkewa.Menene idan abubuwan da aka ƙera ba za su iya guje wa na'urar dimers ba?
Hanyar 3: Rage adadin abubuwan farawa kuma ƙara yawan zafin jiki (babu buƙatar yin bayani).
Matsakaicin ƙimar zafin jiki na annealing shine 60 ° C.Idan ba ku da tabbas, ta yaya za ku zaɓi zafin zafin da ya fi dacewa?Amsar iri ɗaya ce da zaɓin maida hankali na farko -gwajin gradient.Ɗauki hoto daga kamfanin Bio-rad don kwatanta matsalar.Don haɓaka wani yanki mai niyya, saita gradients zafin jiki guda takwas, kowannensu yana da maimaituwa uku, kuma yanayin ƙarawa da aka samu shine kamar haka:

zaɓin zafin jiki mai zafi:
· 70 ° C, 69 ° C - Ainihin, ba za a iya haɗa abubuwan farko ba, don haka babu ƙarawa.
· 67.3 ° C - Akwai ƙananan ƙarawa a farkon, kuma ƙimar Ct yana da girma.
· 64.5°C——Ƙimar Ct tana raguwa.
A 60.7°C, 58.0°C, 56.2°C, da 55.0°C, ƙimar Ct a zahiri tana son zama karko, amma ƙimar haske ta ƙarshe ta bambanta.
Yadda za a zabi?Ƙa'ida: Ƙa'idar farko ita ce ƙimar Ct mafi girma.Don ƙimar Ct iri ɗaya, zaɓi mafi girman zafin jiki mai ɓarna don guje wa raguwa da haɓakawa mara takamaiman.Ko da yake akwai ƙimar haske mafi girma a 55 ° C, ana iya samun dimers ko ƙararrawa marasa takamaiman a ciki.
Amma idan kun kasance masu wayo kamar ku, tabbas za ku yi tunani: A ma'ana, idan amsawar PCR ta keɓantacce, idan dai matakin ƙaddamarwa ya wuce mafi ƙarancin abin da ake buƙata, manyan maki da ƙananan maki bai kamata su yi wani tasiri ba, kamar dyes fluorescent da dNTPs.Lallai, idan dai an inganta yanayin zafin jiki yadda ya kamata, tasirin maida hankali kan ƙimar Ct a zahiri za a rage shi.

An inganta yanayin zafin jiki yadda ya kamata, kuma za a rage tasirin maida hankali kan CT
Tsarin sakandare yana rinjayar ingancin haɓakawa
Bari mu dauki hoto daga Bio-rad don kwatanta matsalar.Hakanan yana ƙirƙira ƙarancin zafin jiki don haɓaka kwayar halitta tare da tsari na biyu.

Tsarin na biyu ya fito
Ana iya ganin cewa yayin da yanayin zafin jiki ya ragu, samfurori sun fara bayyana kuma ƙimar Ct ta ci gaba, ta kai mafi ƙarancin ƙima a 60.7 ° C, sa'an nan kuma yayin da zafin jiki ya ragu, ƙimar Ct ya zama mafi girma.Sabanin haka, yayin da zafin jiki ya karu, tsarin na biyu yana buɗewa kuma haɓakar haɓakawa yana ƙaruwa.Bayan kai wani zazzabi, ƙara yawan zafin jiki ba zai iya inganta haɓakar haɓakawa ba.Domin ba za a iya haɗa fidda kai tsaye a wannan lokacin ba.Don haka,nemo zafin jiki tare da mafi ƙarancin ƙimar Ct, wanda shine mafi kyawun zafin jiki don haɓaka samfurin tsari na biyu!Tabbas, wawaye masu wayo dole ne su san cewa idan ba lallai ba ne, yana da kyau a canza masu farawa kuma ku guje wa yankin tsarin na biyu.
5. matakin aikace-aikace
MIQE-Binciken Bayanai

Ana yin nazarin bayanan ne ta hanyar kayan aikin PCR mai kyalli.A cikin labarin da ya gabata, an yi aikin nazarin bayanai da yawa, irin su kulawar da ba ta dace ba, wanda aka bayyana a cikin ƙirar gwajin.An fayyace nau'ikan kwayoyin halitta na ciki, maimaita lambobi, da sauransu., a nan mun fi bayyana aikace-aikacen qPCR.
qPCR ana amfani da shi sosai, kuma tabbatar da gwaji da tantancewar acid nucleic sune mafi yawan yanayin da ake amfani da su.
cikakken ƙididdigewa
Log (natsuwa na farko) yana da alaƙa ta layi tare da adadin zagayowar.Za a iya zana madaidaicin lanƙwasa daga ma'auni tare da sanannen lambar kwafin farko, wato, ana iya samun alaƙar madaidaiciyar amsawar haɓakawa.Dangane da ƙimar Ct na samfurin, ana iya ƙididdige ƙaddamarwa a cikin samfurin.Adadin samfuran da za a haɗa.

Cikakken Hanyar Ƙididdigar Ƙidaya
Cikakken ƙididdigewa dole ne ya dogara da daidaitaccen lanƙwasa.Don yin daidaitaccen lanƙwasa, ana buƙatar ma'auni.Yawancin lokaci, ma'auni shine plasmid da aka samu ta hanyar cloning gener manufa.Me yasa yake da plasmid?Domin DNA plasmid madauwari shine mafi kwanciyar hankali.Rarraba daidaitaccen samfurin a cikin gradients 5 zuwa 6 bisa ga rabo mai ninki biyu (dilution mai ninki 10), kuma kula da daidaito yayin diluting.Bari ƙimar Ct ta faɗi tsakanin 15-30.

Daidaitaccen shiri
Har ila yau, samfurin da za a gwada ya kamata a diluted daidai (tuna da abin da ake amfani da shi na dilution), kuma darajar Ct kuma ya kamata ya fadi tsakanin 15-30.Daidaitaccen samfurin + samfurin da za a gwada ana saka shi akan injin tare.Bayan gudu, an yi madaidaicin ma'auni tare da daidaitaccen abu, kuma samfurorin da za a gwada an kawo su a cikin ma'auni don ƙididdige ƙaddamarwa.
Hepatitis B kwayar cutar HBV ƙididdigewa ne na yau da kullun cikakkar ƙididdigewa, wanda zai iya ƙididdige lambar kwafin ƙwayoyin cuta a cikin 1ml na jini.
Lissafin lambar kwafi
Samfurin taro da za a gwada (ng/ul) = OD260 × 50ug/ml × dilution factor
Misalin nauyin kwayoyin halitta = adadin tushe × 324
Lambar kwafin samfurin da za a gwada (kwafi / ul) = ƙaddamar da samfurin da za a gwada / nauyin kwayoyin halitta na samfurin × 6 × 1014

Hanyar lissafin kwafin lamba

Abin da ke sama shine hanyar lissafi don ƙayyade adadin.Wannan matsala ce ta ilmin lissafi da za a iya magance ta bayan kammala karatun sakandare, kuma matsalolin ilimin lissafi galibi ana magance su ta hanyar kwamfuta.Idan ba ku gane ba, kuna iya zuwa don sadarwa.
ƙididdigar dangi
An fi amfani da ƙididdige ƙididdigewa a cikin binciken kimiyya.Kwayoyin cuta nawa ne a cikin 1ml na jini, kuma kwayar cutar DNA ce, wannan lamari ne mai ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai: ana iya tantance adadin jinin, kuma kwayar DNA tana da ɗan kwanciyar hankali.Koyaya, yana da wahala a gare mu mu kwatanta adadin kwafin wani nau'in kwayar halitta a cikin ganye, saboda yana da wahala a iya tantance girman, nauyi, da taushin ganyen, adadin RNA da aka cire yana da wuyar tantancewa, kuma ingancin juzu'i shima yana da wuyar tantancewa, wato, kowane mataki na iya sa bayanan gwaji su sami kwari kuma ba za a iya amfani da su ba.
Don haka, ƙayyadaddun dangi dole ne ya gabatar da wani kashi:na ciki reference gene .
A wasu kalmomi, ƙididdige dangi shine ainihin kwatancen tsakanin jigon manufa da kuma na ciki.Idan aka kwatanta a cikin nama iri ɗaya da tantanin halitta iri ɗaya, tasirin girman samfurin, adadin cirewar RNA, jujjuya ingancin rubutu, da ingancin PCR kaɗan ne.Saboda ƙananan girman samfurin, duka kwayoyin halitta na ciki da kuma kwayoyin da aka yi niyya sun ragu sosai.Wannan shine dalilin da ya sa a baya muke jaddada daidaito da kwanciyar hankali.
Ƙwayoyin halittu na ciki gabaɗayakwayoyin halittar gida(Gidan kula da gida), waɗanda ke nufin rukunin kwayoyin halitta waɗanda aka bayyana su a tsaye a cikin dukkan sel, kuma samfuransu suna da mahimmanci don kula da ainihin ayyukan rayuwa na sel.
Kada ku dame wannan ra'ayi.Kwayoyin kiyaye gida sharuɗɗan aikin nazarin halittu ne, yayin da kwayoyin tunani na ciki sharuɗɗan fasaha ne na gwaji.Kwayoyin kula da gida suna buƙatar ƙaddamar da inganci kafin a iya zaɓar su azaman ƙwayoyin tunani na ciki.
Misali, mun zabi kwayoyin halitta da yawa a cikin wannan hoton da ke kasa don gwada matakan bayyanar su a cikin kwayoyin halitta daban-daban, kuma mun gano cewa matakan magana na β-2-microglobulin sun sha bamban da na sauran kwayoyin halitta guda uku, don haka ba za a iya amfani da su azaman kwayoyin tunani na ciki ba.

Bayan fahimtar aikin gyaran gyare-gyare na kwayoyin halitta na ciki, ana samun algorithms guda biyu saboda gabatarwar kwayoyin halitta na ciki.
· Hanyar lankwasa daidaitattun sau biyu
· 2 - △△ Hanyar Ct (Hanyar kwatanta ƙimar CT)
Idan kuna sha'awar nazarin nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan halittu, da fatan za a bar binciken kan algorithms kuma ku yi amfani da dabaru kai tsaye, ko amfani da injina kai tsaye;idan kai mutum ne madaidaiciya a fannin lissafi da injiniyanci, don Allah a ji 'yanci.
Hanyar lankwasa daidaitattun sau biyu
Ƙididdige jigon da aka yi niyya da tsarin kula da gida na samfurin sarrafawa da samfurin da za a gwada ta hanyar ma'auni, sa'an nan kuma ƙididdige ƙimar dangi bisa ga tsarin lissafin, wanda shine matakin magana.
Abũbuwan amfãni: bincike mai sauƙi, ingantaccen gwaji mai sauƙi
Hasara: Ga kowane jinsin halitta, kowane zagaye na gwaje-gwaje dole ne ya yi daidaitaccen lankwasa
Aikace-aikace: Ɗaya daga cikin hanyoyin ƙididdigewa na dangi guda biyu da aka fi amfani da su a cikin nazarin ƙa'idojin bayyana kwayoyin halitta
Tsarin tsari shine kamar haka:

Misalai sune kamar haka:

Yi lissafin adadin dangi bisa sakamakon ƙididdiga
2 - △△ Hanyar Ct (Hanyar kwatanta ƙimar CT)

Abũbuwan amfãni: Babu buƙatar yin daidaitattun lankwasa
Rashin hasara: An ɗauka cewa haɓakar haɓakawa yana kusa da 100%;daidaitattun daidaituwa shine <5%, kuma daidaitaccen ma'auni da inganci tsakanin kowane haɓakawa ana ɗauka ya zama daidai;inganta yanayin gwaji ya fi rikitarwa.
Aikace-aikace: Ɗaya daga cikin hanyoyin ƙididdigewa na dangi guda biyu da aka fi amfani da su a cikin nazarin ƙa'idojin bayyana kwayoyin halitta

Tabbas ingancin ƙarawa yawanci ba zai yuwu ya zama daidai ba 1. Hanyar gyarawa: Idan mun san cewa mahaɗin da aka yi niyya da nau'in kwayar halitta suna da ƙarfin haɓakawa iri ɗaya, amma ƙarfin haɓakawa bai kai 1 ba, to 2－△△ Ct za a iya gyara shi kamar: (1+E )： ： (1+E )－－△ △ △, misali, 5. Ana iya gyara tsarin lissafin zuwa 1.95 - △ △ Ct
Ya zuwa yanzu, abun ciki game da PCR mai kyalli ya zo ƙarshe.