一, Ƙara hankali na tsarin amsawa:

1. Ware babban ingancin RNA:

Nasarar haɗakar cDNA ta fito ne daga RNA mai inganci.RNA mai inganci yakamata ya zama aƙalla ya zama cikakken tsayi kuma ba tare da masu hana masu juyar da rubutu kamar EDTA ko SDS ba.Ingancin RNA yana ƙayyade iyakar adadin bayanan jeri da za ku iya rubutawa zuwa cDNA.Hanyar tsarkakewa ta RNA ta gama gari hanya ce ta mataki ɗaya ta amfani da guanidine isothiocyanate/acid phenol.Don hana kamuwa da cuta ta hanyar gano adadin RNase, RNA keɓe daga samfuran RNase masu wadatar (kamar pancreas) yana buƙatar adana shi a cikin formaldehyde don adana RNA mai inganci, musamman don adana dogon lokaci.RNA da aka ciro daga hantar bera ta ragu sosai bayan an adana shi a cikin ruwa har tsawon mako guda, yayin da RNA da aka ciro daga hanjin bera ta kasance a kwance bayan an adana shi cikin ruwa tsawon shekaru 3.Bugu da kari, kwafi fiye da 4kb sun fi kula da lalacewa ta hanyar RNases fiye da ƙananan kwafi.Don ƙara kwanciyar hankali na samfuran RNA da aka adana, ana iya narkar da RNA a cikin nau'in nau'in deionized kuma a adana shi a -70°C.Formamide da ake amfani da shi don adana RNA dole ne ya zama mara amfani da tarkace mai lalata RNA.Ana iya adana RNA daga pancreas a cikin foramide na akalla shekara guda.Lokacin shirya amfani da RNA, zaku iya amfani da hanyar da ke gaba don haɓaka RNA: ƙara NaCl zuwa 0.2M da ƙarar ethanol sau 4, sanya a cikin ɗaki na minti 3-5, da centrifuge a 10,000 × g na mintuna 5.

2. Yi amfani da rubutun baya na RNaseH (RNaseH-):

Ana ƙara masu hana RNase sau da yawa don juyar da halayen rubutun don ƙara tsayi da yawan amfanin haɗin cDNA.Ya kamata a ƙara masu hana RNase a lokacin haɗin haɗin farko na farko a gaban mai ɗaukar hoto da wakili mai ragewa (kamar DTT), saboda tsarin da aka yi kafin haɗin cDNA yana hana mai hanawa, don haka yana sakin RNase mai ɗaure wanda zai iya lalata RNA.Protein RNase inhibitors kawai suna hana lalata RNA ta hanyar RNase A, B, C, kuma ba sa hana RNase akan fata, don haka a kula kar a gabatar da RNase daga yatsun ku duk da amfani da waɗannan masu hanawa.

Reverse transcriptase yana haifar da juyar da RNA zuwa cDNA.Dukansu M-MLV da AMV suna da ayyukan RNaseH na ƙarshe ban da nasu ayyukan polymerase.Ayyukan RNaseH da ayyukan polymerase suna gasa da juna don 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 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) na DNA ko cDNA.Samfurin RNA da ayyukan RNaseH ya ƙasƙanta ba zai iya zama madaidaicin madauri don haɗin cDNA ba, wanda ke rage yawan amfanin ƙasa da tsawon haɗin cDNA.Don haka, zai zama da fa'ida don kawar da ko rage yawan ayyukan RNaseH na juzu'i.

SuperScript.Adadin haɗin cDNA zai shafe hankalin RT-PCR.ThermoScript ya fi AMV kulawa sosai.Girman samfuran RT-PCR yana iyakance ta ikon jujjuya rubutun don haɗa cDNA, musamman lokacin rufe manyan cDNAs.Idan aka kwatanta da MMLV, SuperScripⅡ yana haɓaka yawan amfanin dogayen samfuran RT-PCR.Har ila yau, RNaseH-reverse transcriptase ya ƙara yawan zafin jiki, don haka ana iya aiwatar da martani a yanayin zafi sama da na al'ada 37-42°C.A ƙarƙashin sharuɗɗan haɗakar da aka ba da shawarar, yi amfani da firamare oligo(dT) da 10 μCi na [α-P] dCTP.An ƙididdige jimlar yawan amfanin ƙasa na farko ta amfani da hanyar hazo TCA.An yi nazarin cDNA mai cikakken tsayi ta hanyar amfani da madaukai masu girma da aka cire kuma an ƙidaya su akan gel na agarose na alkaline.

3. Haɓaka zafin jiki don juyar da rubutu:

Matsayin zafin jiki mafi girma yana taimakawa buɗe tsarin na biyu na RNA, yana ƙara yawan abin da ake samu.Ga mafi yawan samfuran RNA, sanya RNA da abubuwan farko a 65°C ba tare da buffer ko gishiri ba, tare da saurin sanyaya kan kankara zai kawar da yawancin tsarin na biyu kuma ya ba da izinin ɗauri.Koyaya, wasu samfuran har yanzu suna da sifofi na biyu, ko da bayan ƙarancin zafi.Ana iya ƙara haɓaka waɗannan samfuran masu wahala ta amfani da ThermoScript Reverse Transcriptase da sanya jujjuyawar juzu'i a yanayin zafi mafi girma don haɓaka haɓakawa.Hakanan yanayin zafi mafi girma na iya ƙara ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, musamman lokacin da aka yi amfani da ƙayyadaddun ƙayyadaddun ƙirar halitta (GSP) don haɗin cDNA (duba Babi na 3).Idan ana amfani da GSP, tabbatar da cewa Tm na firamare daidai yake da yanayin da ake tsammani.Kada a yi amfani da oligo(dT) da bazuwar firamare sama da 60°C.Matsakaicin bazuwar suna buƙatar shiryawa a 25 ° C na minti 10 kafin ƙarawa zuwa 60 ° C.Baya ga yin amfani da mafi girman juzu'in juzu'i, ana iya haɓaka ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadad da su ke canja wurin RNA/primer gauraya kai tsaye daga zafin zafi na 65°C zuwa yanayin yanayin juzu'i na juye-juye da ƙara cakudewar amsawar 2 × pre-warmed (cDNA hot-fara kira) .Wannan hanya tana taimakawa hana haɗin haɗin ginin tsakanin kwayoyin halitta wanda ke faruwa a ƙananan yanayin zafi.Ana iya sauƙaƙa da sauyawar zafin jiki da yawa don RT-PCR ta amfani da na'ura mai zafi.

polymerase Tth thermostable yana aiki azaman DNA polymerase a gaban Mg2+ kuma azaman RNA polymerase a gaban Mn2+.Ana iya kiyaye shi da dumi a iyakar zafin jiki na 65 ° C.Koyaya, kasancewar Mn2+ yayin PCR yana rage aminci, wanda ke sa Tth polymerase ya zama ƙasa da dacewa don haɓaka daidaitattun daidaito, kamar cloning na cDNA.Bugu da ƙari, Tth yana da ƙananan juzu'i na juzu'i, wanda ke rage hankali, kuma, tun da juzu'in juzu'i da PCR za a iya yi tare da enzyme guda ɗaya, ba za a iya amfani da halayen sarrafawa ba tare da juzu'i na juzu'i ba don kwatanta samfuran haɓakawa na cDNA tare da gurɓataccen DNA na genomic.An raba samfuran haɓakawa.

4. Abubuwan da ke inganta rubutun baya:

Additives ciki har da glycerol da DMSO ana kara su zuwa aikin haɗin gwiwar farko, wanda zai iya rage kwanciyar hankali na nucleic acid sau biyu kuma ya kwance tsarin na biyu na RNA.Ana iya ƙara har zuwa 20% glycerol ko 10% DMSO ba tare da shafar ayyukan SuperScript II ko MMLV ba.AMV kuma na iya jurewa har zuwa 20% glycerol ba tare da asarar aiki ba.Don haɓaka hankalin RT-PCR a cikin SuperScriptⅡ amsawar juyar da rubutu, ana iya ƙara 10% glycerol kuma a sanya shi a 45°C.Idan an ƙara 1/10 na samfurin amsawar juzu'i zuwa PCR, to ƙimar glycerol a cikin haɓakar haɓakawa shine 0.4%, wanda bai isa ya hana PCR ba.

5. Maganin RNaseH:

Jiyya na halayen haɗin cDNA tare da RNaseH kafin PCR na iya ƙara hankali.Ga wasu samfura, ana tunanin cewa RNA a cikin haɗin haɗin cDNA yana hana ɗaurin samfuran haɓakawa, wanda a cikin yanayin jiyya na RNaseH na iya haɓaka hankali.Gabaɗaya, jiyya na RNaseH ya zama dole lokacin haɓaka samfuran cDNA mai tsayi mai tsayi, kamar ƙananan kwafin tuberous scherosis II.Don wannan samfuri mai wahala, jiyya na RNaseH ya haɓaka siginar da SuperScript II ko cDNA da aka haɗa AMV ke samarwa.Ga mafi yawan halayen RT-PCR, magani na RNaseH zaɓi ne, saboda matakin denaturation na PCR a 95°C gabaɗaya yana haɓaka RNA a cikin hadaddun RNA:DNA.

6. Inganta Ƙaramar Hanyar Ganewar RNA:

RT-PCR yana da ƙalubale musamman lokacin da ƙananan adadin RNA ke samuwa.Glycogen da aka ƙara azaman mai ɗaukar hoto yayin keɓewar RNA yana taimakawa wajen haɓaka yawan samfuran ƙananan samfuran.Ana iya ƙara glycogen marasa RNase a lokaci guda tare da ƙara Trizol.Glycogen shine mai narkewar ruwa kuma ana iya adana shi a cikin ruwa mai ruwa tare da RNA don taimakawa hazo mai zuwa.Don samfuran ƙasa da 50 MG na nama ko sel na al'ada 106, shawarar da aka ba da shawarar na glycogen mara amfani da RNase shine 250 μg/ml.

Ƙara BSA acetylated zuwa juyar da juzu'i ta amfani da SuperScript II na iya ƙara azanci, kuma ga ƙananan adadin RNA, rage adadin SuperScript II da ƙara raka'a 40 na RNaseOut nuclease inhibitor na iya ƙara matakin ganowa.Idan ana amfani da glycogen a cikin tsarin keɓewar RNA, ana ba da shawarar ƙara BSA ko RNase inhibitor yayin amfani da SuperScript II don mayar da martanin rubutu.

二, Ƙara takamaiman RT-PCR

1. CND Asynthesis:

Za'a iya ƙaddamar da haɗin cDNA na farko ta amfani da hanyoyi daban-daban guda uku, ƙayyadaddun ƙayyadaddun dangi wanda ke rinjayar adadin da nau'in cDNA da aka haɗa.

Hanyar farko ta bazuwar ita ce mafi ƙayyadaddun ƙayyadaddun hanyoyin guda uku.Mahimman bayanai suna buɗewa a shafuka da yawa a duk tsawon rubutun, suna haifar da gajeriyar cDNAs mai tsayi-tsayi.Ana amfani da wannan hanyar akai-akai don samun jerin ƙarshen 5′ kuma don samun cDNA daga samfuran RNA tare da yankuna na tsarin sakandare ko tare da wuraren ƙarewa waɗanda ba za a iya kwafi su ta hanyar juyar da rubutu ba.Don samun mafi tsayin cDNA, ana buƙatar ƙaddara rabon firamare zuwa RNA a cikin kowane samfurin RNA a zahiri.Matsakaicin farawa na abubuwan da bazuwar bazuwar ya kasance daga 50 zuwa 250 ng a cikin amsawar 20 μl.Tunda cDNA da aka haɗa daga jimillar RNA ta amfani da bazuwar firamare shine RNA ribosomal, gabaɗaya ana zaɓar poly(A)+RNA azaman samfuri.

Alamar Oligo(dT) sun fi ƙayyadaddun ƙayyadaddun ƙayyadaddun abubuwa.Yana haɓaka zuwa jelar poly(A) da aka samo a ƙarshen 3′ mafi yawan eukaryotic mRNAs.Saboda poly(A)+ RNA shine kusan 1% zuwa 2% na jimillar RNA, adadin da sarƙaƙƙiyar cDNA ya yi ƙasa da na bazuwar firamare.Saboda ƙayyadaddun ƙayyadaddun sa, oligo(dT) gabaɗaya baya buƙatar haɓaka ƙimar RNA zuwa abubuwan da aka tsara da zaɓin poly(A)+.Ana ba da shawarar yin amfani da 0.5μg oligo(dT) akan tsarin amsawa na 20μl.oligo(dT)12-18 ya dace da yawancin RT-PCR.Tsarin ThermoScript RT-PCR yana ba da oligo(dT)20 saboda mafi kyawun yanayin yanayin zafi don haɓakar yanayin zafi.

Gene Specific primers (GSP) su ne mafi ƙayyadaddun firikwensin don matakin juyar da rubutu.GSP shine oligonucleotide na antisense wanda zai iya haɓaka musamman zuwa jerin maƙasudin RNA, sabanin bazuwar al'ada ko oligo(dT), waɗanda ke lalata duk RNAs.Irin waɗannan ƙa'idodin da aka yi amfani da su don ƙirƙira abubuwan farko na PCR sun shafi ƙirar GSP a jujjuya halayen rubutu.GSP na iya zama jeri iri ɗaya da na'urar ƙarawa wanda ke buɗewa zuwa 3'-mafi yawan ƙarshen mRNA, ko kuma ana iya ƙirƙira GSP don rusa ƙasa na maɓallin ƙarawa na baya.Ga wasu batutuwan da aka haɓaka, ana buƙatar ƙirƙira firamare na antisense fiye da ɗaya don samun nasara na RT-PCR saboda tsarin na biyu na RNA da aka yi niyya na iya hana ɗaurin firam.Ana ba da shawarar yin amfani da 1 pmol antisense GSP a cikin 20 μl na haɗin haɗin farko.

2. Haɓaka zafin jiki don juyar da rubutu:

Domin samun cikakkiyar fa'idar takamaiman GSP, yakamata a yi amfani da juzu'i na juzu'i tare da mafi girman yanayin zafi.Za'a iya shigar da bayanan juzu'i na thermostable a yanayin zafi mafi girma don ƙara ƙarfin amsawa.Misali, idan GSP annes a 55°C, ƙayyadaddun GSP ba za a yi cikakken amfani da shi ba idan aka yi amfani da AMV ko M-MLV don juyar da rubutu a ƙaramin ƙarfi na 37°C.Koyaya, SuperScript II da ThermoScript za a iya mayar da martani a 50°C ko sama, wanda zai kawar da takamaiman samfuran da aka samar a ƙananan yanayin zafi.Don ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, ana iya canja wurin hadawar RNA/primer kai tsaye daga zafin 65°C denaturation zuwa yanayin juzu'i na jujjuyawar rubutu kuma ƙara zuwa gauran amsawar 2 × da aka riga aka yi dumi (ƙarfin cDNA mai zafi mai zafi).Wannan yana taimakawa hana haɗin tushe na intermolecular a ƙananan yanayin zafi.Ana iya sauƙaƙa canjin yanayin zafi da yawa da ake buƙata don RT-PCR ta amfani da na'urar hawan zafi.

3. Yana rage gurɓatar halittar DNA:

Matsala mai yuwuwar ci karo da RT-PCR shine gurɓatar halittar DNA a cikin RNA.Yin amfani da kyakkyawar hanyar keɓewar RNA, kamar Trizol Reagent, zai rage adadin DNA na genomic da ke gurɓata shirye-shiryen RNA.Don guje wa samfuran da aka samo daga DNA na genomic, ana iya bi da RNA tare da ƙaramar darajar DNAse I don cire gurɓataccen DNA kafin a juyar da rubutu.An ƙare narkewar DNase I ta hanyar haɗa samfuran a cikin 2.0 mM EDTA na mintuna 10 a 65°C.EDTA na iya chelate magnesium ions, hana magnesium ion dogara RNA hydrolysis a babban yanayin zafi.

Domin raba cDNA da aka haɓaka daga gurɓatar samfuran ƙarar DNA na genomic, ana iya tsara abubuwan da za a iya ƙirƙira waɗanda kowane keɓancewa don raba exons.Kayayyakin PCR da aka samu daga cDNA za su yi guntu fiye da waɗanda aka samu daga gurɓataccen DNA na genomic.Bugu da ƙari, an yi gwajin sarrafawa ba tare da jujjuya rubutu ba akan kowane samfurin RNA don tantance ko an samo guntuwar da aka bayar daga DNA na genomic ko cDNA.Samfurin PCR da aka samu ba tare da juyar da rubutu ba an samo shi daga kwayoyin halitta.