An haɓaka RT-qPCR daga fasahar PCR na yau da kullun.Yana ƙara sinadarai masu kyalli ( rini mai kyalli ko bincike mai kyalli) zuwa tsarin amsawar PCR na al'ada, kuma yana gano tsarin cirewa da haɓakawa na PCR a ainihin lokacin bisa ga hanyoyin haskensu daban-daban.Ana amfani da canje-canjen siginar mai walƙiya a cikin matsakaici don ƙididdige adadin canjin samfur a kowane zagaye na PCR.A halin yanzu, hanyoyin da aka fi sani sune hanyar rini mai kyalli da hanyar bincike.

Hanyar rini mai walƙiya:
Wasu rini mai kyalli, irin su SYBR Green Ⅰ, PicoGreen, BEBO, da sauransu, ba sa fitar da haske da kansu, amma suna fitar da haske bayan sun ɗaure zuwa ƙaramin tsagi na dsDNA.Sabili da haka, a farkon amsawar PCR, injin ba zai iya gano siginar kyalli ba.Lokacin da martani ya ci gaba zuwa tsawaita-tsawo (hanyar mataki biyu) ko matakin tsawo (hanyar mataki uku), ana buɗe igiyoyi biyu a wannan lokacin, da sabon DNA polymerase A lokacin haɗin igiyoyi, ƙwayoyin fluorescent suna haɗuwa a cikin ƙaramin tsagi na dsDNA kuma suna fitar da haske.Yayin da adadin zagayowar PCR ya karu, rinai da yawa suna haɗuwa da dsDNA, kuma siginar kyalli kuma ana ci gaba da haɓakawa.Dauki SYBR Green Ⅰ a matsayin misali.
Hanyar bincike:
Binciken Taqman shine binciken hydrolysis da aka fi amfani dashi.Akwai rukuni mai kyalli a ƙarshen 5′ na binciken, yawanci FAM.Binciken kansa jeri ne mai dacewa da kwayar halittar da aka yi niyya.Akwai ƙungiyar kashe kyalli a ƙarshen 3′ na fluorophore.Dangane da ka'idar Canjawar Canjin Canjin Canjin Canjin Canjin Canja wurin (Förster Resonance Energy Transfer, FRET), lokacin da ƙungiyar mai ba da labari mai kyalli (mai ba da gudummawa mai kyalli) da ƙungiyar mai kyalli mai kyalli (ƙwayar kyalli mai karɓa) Lokacin da bakan bakan ya mamaye kuma nesa yana kusa sosai (7-10nm na iya ba da izinin iskar gas). 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.

Hoto na 1 Ƙa'idar qRT-PCR

Zane na farko
Ka'idoji:

Ya kamata a tsara abubuwan farko a cikin yankin da aka adana na jerin nucleic acid kuma suna da takamaiman.

Zai fi kyau a yi amfani da jerin cDNA, kuma jerin mRNA kuma abin karɓa ne.Idan ba haka ba, gano tsarin yanki na cds na jerin DNA.
Tsawon samfurin ƙididdigewa shine 80-150bp, mafi tsayi shine 300bp, tsayin farko shine gabaɗaya tsakanin sansanonin 17-25, kuma bambancin da ke tsakanin sama da na ƙasa bai kamata ya zama babba ba.

Abubuwan G+C suna tsakanin 40% da 60%, kuma 45-55% shine mafi kyau.
Ƙimar TM tana tsakanin digiri 58-62.
Yi ƙoƙarin kauce wa dimers na farko da dimers na kai, (kada ku bayyana fiye da nau'i-nau'i 4 na madaidaicin madaidaicin tushe) tsarin gashin gashi, idan ba za a iya kauce masa ba, yi ΔG <4.5kJ / mol * Idan ba za ku iya tabbatar da cewa an cire gDNA a lokacin rubutun baya Tsabtace, yana da kyau a tsara abubuwan da aka tsara na intron, ba za a iya gyara G / G ba, da kuma guje wa yankunan da ba za a iya gyarawa ba, G AT / G, don kauce wa yankunan da ba za a iya gyarawa ba. Tsari mai ci gaba (2-3) masu farawa da waɗanda ba
ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai ya fi dacewa ƙasa da 70% ko kuma yana da 8 ƙarin ƙayyadaddun homology.
Database:
Neman audugaFGD ta kalmomi masu mahimmanci
Zane na farko:
IDT-qPCR ƙira

Fig2 IDT kan layi kayan aikin ƙirar kayan aikin farko

Hoton sakamako na Fig3 nuni
Zane na abubuwan farko na lncRNA:
lncRNA:matakai iri ɗaya da mRNA.
miRNA:Ka'idar hanyar madauki mai tushe: Tunda duk miRNAs gajeru ne na kusan 23 nt, ba za a iya gano PCR kai tsaye ba, don haka ana amfani da kayan aikin jeri-madauki.Tsarin madauki mai tushe shine DNA mai ɗauri ɗaya na kusan 50 nt, wanda zai iya samar da tsarin gashin gashi da kanta.3 'Ana iya ƙirƙira ƙarshen azaman jeri mai dacewa da juzu'in miRNA, sannan ana iya haɗa miRNA manufa zuwa jerin madauki yayin jujjuyawar, kuma jimlar tsayin zai iya kaiwa 70bp, wanda yayi daidai da tsayin samfurin haɓakawa wanda qPCR ya ƙaddara.Tailing miRNA primer zane.
Takamaiman gano haɓakawa:
Bayanai na fashewar kan layi: fashewar CottonFGD ta hanyar kamanceceniya
Ƙwaƙwalwar gida: Koma zuwa amfani da Blast + don yin fashewar gida, Linux da macos na iya kafa bayanan gida kai tsaye, tsarin win10 kuma ana iya yin shi bayan shigar da ubuntu bash.Ƙirƙirar bayanan fashewar gida da fashewar gida;Bude ubuntu bash akan win10.
Sanarwa: Auduga na sama da audugar tsibiri na tetraploid amfanin gona ne, don haka sakamakon fashewar zai kasance sau biyu ko fiye da ashana.A baya, yin amfani da NAU cds a matsayin ma'ajin bayanai don yin fashewa mai yuwuwa nemo kwayoyin halitta guda biyu masu kama da 'yan bambance-bambancen SNP.Yawancin lokaci, kwayoyin halitta guda biyu ba za a iya raba su ta hanyar zane na farko ba, don haka ana kula da su iri ɗaya.Idan akwai bayyananniyar indel, yawanci ana tsara firam ɗin akan indel, amma wannan na iya haifar da tsarin na biyu na firamare Ƙarfin kuzarin ya zama mafi girma, yana haifar da raguwar haɓakar haɓakawa, amma wannan ba shi yiwuwa.

Gano tsarin farko na sakandare:
Matakai:bude oligo 7 → jerin samfurin shigarwa → kusa da taga sub-taga → ajiyewa → gano wuri a kan samfuri, latsa ctrl+D don saita tsawon lokaci → nazarin sassa daban-daban na sakandare, kamar jiki mai sarrafa kansa, heterodimer, hairpin, mismatch, da dai sauransu. Hotuna biyu na karshe a cikin Hoto 4 sune sakamakon gwaji na masu farawa.Sakamakon farko na gaba yana da kyau, babu wani dimer mai mahimmanci da tsarin gashin gashi, babu ci gaba da tushe mai mahimmanci, kuma cikakkiyar darajar makamashi na kyauta ya kasance ƙasa da 4.5, yayin da na baya na baya ya nuna ci gaba da 6 tushe ne mai dacewa, kuma makamashi na kyauta shine 8.8;Bugu da kari, dimer mafi tsanani yana bayyana a ƙarshen 3′, kuma dimer na sansanoni 4 a jere ya bayyana.Kodayake makamashin kyauta ba shi da girma, 3′ dimer Chl na iya yin tasiri sosai ga ƙayyadaddun haɓakawa da haɓakar haɓakawa.Bugu da ƙari, wajibi ne don bincika gashin gashi, heterodimers, da rashin daidaituwa.

Fig3 oligo7 sakamakon ganowa
Gano ingantaccen haɓakawa:
Ingantacciyar haɓakawa na halayen PCR yana tasiri sosai ga sakamakon PCR.Hakanan a cikin qRT-PCR, haɓakar haɓakawa yana da mahimmanci musamman ga sakamakon ƙididdiga.Cire wasu abubuwa, injuna da ladabi a cikin ma'ajin amsawa.Har ila yau, ingancin firamare yana da babban tasiri akan ingancin haɓakawa na qRT-PCR.Domin tabbatar da daidaiton sakamakon, duka ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun haske da cikakkiyar ƙididdigewa suna buƙatar gano ingancin haɓakawa na firamare.An gane cewa ingantacciyar qRT-PCR haɓaka haɓakawa tana tsakanin 85% da 115%.Akwai hanyoyi guda biyu:
1. Daidaitaccen Hanyar lankwasa:
a.Mix cDNA
b.Dilution na gradient
c.qPCR
d.Ma'aunin koma baya na layi don ƙididdige ingancin haɓakawa
2. LinRegPCR
LinRegPCR shiri ne don nazarin bayanan RT-PCR na ainihin lokaci, wanda kuma ake kira ƙididdigar PCR (qPCR) data dangane da SYBR Green ko makamancinsu.Shirin yana amfani da bayanan gyare-gyaren da ba na asali ba, yana yin gyare-gyare na asali akan kowane samfurin Daban-daban, ƙayyade taga-linearity sa'an nan kuma yayi amfani da bincike na linzamin kwamfuta don dacewa da madaidaiciyar layi ta hanyar saitin bayanan PCR.Daga gangaren wannan layin ana ƙididdige ingancin PCR na kowane samfuri.Ana amfani da ma'anar ƙimar PCR a kowane amplicon da ƙimar Ct kowane samfurin don ƙididdige ƙaddamarwar farawa kowane samfurin, wanda aka bayyana a cikin raka'a mai walƙiya na sabani.Shigar da bayanai da fitarwa suna ta hanyar maƙunsar bayanai na Excel.Samfura kawai
ana buƙatar hadawa, babu gradient
ana buƙatar matakai:(Ɗauki Bole CFX96 a matsayin misali, ba injina ba tare da bayyananniyar ABI)
gwaji:gwajin qPCR daidai ne.
fitar da bayanan qPCR:LinRegPCR na iya gane nau'ikan fayilolin fitarwa guda biyu: RDML ko sakamakon Amplification.A haƙiƙa, ƙimar gano ainihin lokacin lambar sake zagayowar da siginar kyalli ta na'ura, kuma ana samun haɓakawa ta hanyar nazarin ƙimar canjin haske na ingancin sashin layi.
Zaɓin bayanai: A ka'idar, ƙimar RDML yakamata ta kasance mai amfani.An kiyasta cewa matsalar kwamfuta ta ita ce software ba za ta iya gane RDML ba, don haka ina da ƙimar fitarwa ta excel a matsayin ainihin bayanan.Ana ba da shawarar yin wani m nunin bayanai na farko, kamar gazawar ƙara samfurori, da dai sauransu. Ana iya share maki a cikin bayanan fitarwa (ba shakka, ba za ku iya share su ba, LinRegPCR zai yi watsi da waɗannan maki a cikin mataki na gaba).

Fig5 qPCR bayanan fitarwa

Fig6 zaɓin samfuran ɗan takara

Shigar da bayanai:Buɗe sakamakon haɓaka cancantar.xls, → buɗe LinRegPCR → fayil → karantawa daga excel → zaɓi sigogi kamar yadda aka nuna a Hoto 7 → Ok → danna ƙayyade tushen layi

Matakan Fig7 na shigar da bayanan linRegPCR

Sakamako:Idan babu maimaituwa, ba a buƙatar haɗawa.Idan akwai maimaitawa, za a iya gyara rukunin a cikin rukunin samfurin, sannan a shigar da sunan kwayar halittar a cikin mai ganowa, sa'an nan kuma za a haɗa nau'ikan wannan ta atomatik.A ƙarshe, danna fayil ɗin, fitarwa Excel, kuma duba sakamakon.Za a nuna ingancin haɓakawa da sakamakon R2 na kowace rijiya.Abu na biyu, idan kun rarraba zuwa ƙungiyoyi, za a nuna matsakaicin matsakaicin ƙarfin haɓakawa.Tabbatar cewa ingancin haɓakawa na kowane firamare yana tsakanin 85% da 115%.Idan girmansa ya yi yawa ko ƙanƙanta, yana nufin cewa haɓakar haɓakawa na firamare ba shi da kyau.

Hoto na 8 Sakamakon da fitar da bayanai

Tsarin gwaji:
Bukatun ingancin RNA:
Tsafta:1.72.0 yana nuna cewa ana iya samun ragowar isothiocyanate.Tsabtace acid nucleic A260 / A230 ya kamata ya kasance a kusa da 2. Idan akwai karfi mai karfi a 230 nm, yana nuna cewa akwai kwayoyin halitta irin su phenate ions.Bugu da ƙari, ana iya gano shi ta hanyar 1.5% agarose gel electrophoresis.Nuna alamar, saboda ssRNA ba ta da ƙima kuma logarithm ɗin nauyin kwayoyin halitta ba shi da alaƙar layi, kuma ba za a iya bayyana nauyin kwayoyin daidai ba.Hankali: A ka'idabakasa da 100ng/ul, idan maida hankali ya yi ƙasa da ƙasa, tsafta gabaɗaya ba ta da tsayi

9 RNA gel

Bugu da kari, idan samfurin yana da daraja kuma tarin RNA yana da girma, ana ba da shawarar a raba shi bayan an cire shi, kuma a tsoma RNA zuwa matakin ƙarshe na 100-300ng/ul don juyar da rubutu.A cikitsarin juyar da rubutu, lokacin da aka rubuta mRNA, oligo (dt) masu mahimmanci waɗanda za su iya ɗaure musamman ga wutsiyar polyA ana amfani da su don juyar da rubutu, yayin da lncRNA da circRNA suna amfani da bazuwar hexamer (Random 6 mer) don juyar da rubutun jimlar RNA Ga miRNA, miRNA-musamman madaidaicin madauki na madauki na miRNA ana amfani da shi don juyawa.Kamfanoni da yawa yanzu sun ƙaddamar da kayan aikin wutsiya na musamman.Don hanyar madauki, hanyar wutsiya ta fi dacewa, babban kayan aiki, da ceton reagent, amma tasirin bambance miRNA na dangi ɗaya bai kamata ya yi kyau kamar hanyar madauki ba.Kowane juzu'i na juzu'i yana da buƙatu don tattara takamaiman ƙayyadaddun ƙayyadaddun kwayoyin halitta (stem- madaukai).Bayanan ciki da aka yi amfani da shi don miRNA shine U6.A cikin tsarin jujjuyawar madauki mai tushe, bututu na U6 yakamata a jujjuya shi daban, kuma a ƙara abubuwan gaba da baya na U6 kai tsaye.Dukansu circRNA da lncRNA na iya amfani da HKGs azaman tunani na ciki.A cikigano cDNA,
idan babu matsala tare da RNA, cDNA shima yakamata yayi kyau.Koyaya, idan ana bin kamalar gwajin, yana da kyau a yi amfani da jigon tunani na ciki (Reference gene, RG) wanda zai iya bambanta gDNA daga cds.Gabaɗaya, RG gene ce ta kiyaye gida., HKG) kamar yadda aka nuna a hoto na 10;A lokacin, ina yin furotin na ajiyar waken soya, kuma na yi amfani da actin7 mai ɗauke da introns azaman bayanin ciki.Girman ɓangarorin da aka haɓaka na wannan firamare a gDNA ya kasance 452bp, kuma idan an yi amfani da cDNA azaman samfuri, ya kasance 142bp.Sa'an nan sakamakon gwajin ya gano cewa wani ɓangare na cDNA ya gurɓata da gDNA, kuma ya tabbatar da cewa babu matsala game da sakamakon juzu'i, kuma ana iya amfani da shi azaman samfuri na PCR.Ba shi da amfani don gudu agarose gel electrophoresis kai tsaye tare da cDNA, kuma bandeji ne mai yaduwa, wanda ba shi da tabbas.

Hoto 10 gano cDNA

Ƙaddamar da yanayin qPCRGabaɗaya ba matsala bisa ƙa'idar kit ɗin, galibi a matakin ƙimar tm.Idan wasu na'urorin ba a tsara su da kyau ba yayin ƙirar ƙirar ƙira, wanda ke haifar da babban bambanci tsakanin ƙimar tm da ka'idar 60 ° C, ana ba da shawarar cewa cDNA Bayan an haɗu da samfuran, gudanar da PCR mai gradient tare da masu haɓakawa, kuma kuyi ƙoƙarin guje wa saita zafin jiki ba tare da bandeji azaman ƙimar TM ba.

Binciken bayanai

Hanyar sarrafa fluorescence na al'ada na yau da kullun PCR shine ainihin bisa ga 2- ΔΔCT.Samfurin sarrafa bayanai.

Samfura masu dangantaka:

Real Time PCR Sauƙi^TM – Taqman

Real Time PCR Sauƙi^TM -SYBR GREEN I

RT Easy I (Master Premix don haɗin cDNA na farko)

RT Easy II (Master Premix don haɗin cDNA na farko don qPCR)