一、Kuwonjezera chidwi cha machitidwe amachitidwe:

1. Dzipatulani RNA yapamwamba kwambiri:

Kuphatikizika kwa cDNA kopambana kumachokera ku RNA yapamwamba kwambiri.RNA yapamwamba kwambiri iyenera kukhala yayitali komanso yopanda zoletsa reverse transcriptase monga EDTA kapena SDS.Ubwino wa RNA umatsimikizira kuchuluka kwazomwe mungalembe mu cDNA.Njira yodziwika bwino yoyeretsera RNA ndi njira imodzi yokha yogwiritsira ntchito guanidine isothiocyanate/acid phenol.Pofuna kupewa kuipitsidwa ndi kuchuluka kwa RNase, RNA yotalikirana ndi zitsanzo za RNase (monga kapamba) iyenera kusungidwa mu formaldehyde kuti isunge RNA yapamwamba kwambiri, makamaka kuti isungidwe kwa nthawi yayitali.RNA yotengedwa ku chiwindi cha makoswe idawonongeka pambuyo posungidwa m'madzi kwa sabata imodzi, pomwe RNA yotengedwa ku ndulu ya makoswe idakhalabe yokhazikika itasungidwa m'madzi kwa zaka zitatu.Kuphatikiza apo, zolembedwa zazitali kuposa 4 kb zimakhudzidwa kwambiri ndi kuwonongeka ndi trace RNases kuposa zolemba zazing'ono.Kuonjezera kukhazikika kwa zitsanzo za RNA zosungidwa, RNA ikhoza kusungunuka mu deionized formamide ndikusungidwa pa -70 ° C.Formamide yomwe imagwiritsidwa ntchito posunga RNA iyenera kukhala yopanda zinyalala zowononga RNA.RNA yochokera ku kapamba imatha kusungidwa mu formamide kwa chaka chimodzi.Pokonzekera ntchito RNA, mungagwiritse ntchito njira zotsatirazi precipitate RNA: kuwonjezera NaCl kuti 0,2M ndi 4 nthawi buku la Mowa, malo firiji kwa mphindi 3-5, ndi centrifuge pa 10,000 × g kwa mphindi 5.

2. Gwiritsani ntchito RNaseH-inactive (RNaseH-) reverse transcriptase:

Ma RNase inhibitors nthawi zambiri amawonjezedwa kuti asinthe kalembedwe kuti awonjezere kutalika ndi zokolola za kaphatikizidwe ka cDNA.RNase inhibitors ayenera kuwonjezeredwa panthawi ya kaphatikizidwe ka chingwe choyamba pamaso pa chotchinga ndi chochepetsera (monga DTT), chifukwa ndondomeko isanafike cDNA synthesis denatures inhibitor, potero imamasula RNase yomangidwa yomwe ingawononge RNA.Mapuloteni a RNase inhibitors amangolepheretsa kuwonongeka kwa RNA ndi RNase A, B, C, ndipo samalepheretsa RNase pakhungu, choncho samalani kuti musalowetse RNase kuchokera ku zala zanu ngakhale mutagwiritsa ntchito izi.

Reverse transcriptase imathandizira kutembenuka kwa RNA kukhala cDNA.Onse M-MLV ndi AMV ali ndi endogenous RNaseH zochita kuwonjezera pa polymerase zochita zawo.Zochita za RNaseH ndi zochitika za polymerase zimapikisana wina ndi mnzake pa chingwe chosakanizidwa chomwe chimapangidwa pakati pa template ya RNA ndi DNA primer kapena cDNA extension strand, ndikunyozetsa chingwe cha RNA mu RNA:DNA complex.The RNA template yodetsedwa ndi ntchito ya RNaseH singakhalenso gawo lothandiza la kaphatikizidwe ka cDNA, zomwe zimachepetsa zokolola ndi kutalika kwa kaphatikizidwe ka cDNA.Chifukwa chake, zingakhale zopindulitsa kuthetsa kapena kuchepetsa kwambiri ntchito ya RNaseH ya reverse transcriptase..

SuperScript Ⅱ reverse transcriptase, RNaseH- MMLV reverse transcriptase ndi thermoScript reverse transcriptase, RNaseH- AMV, imatha kupeza kuchuluka komanso kutalika kwa cDNA kuposa MMLV ndi AMV.Kukhudzidwa kwa RT-PCR kudzakhudzidwa ndi kuchuluka kwa kaphatikizidwe ka cDNA.ThermoScript ndizovuta kwambiri kuposa AMV.Kukula kwa zinthu za RT-PCR kumachepa chifukwa cha kuthekera kwa reverse transcriptase kupanga cDNA, makamaka popanga ma cDNA akulu.Poyerekeza ndi MMLV, SuperScripⅡ idakulitsa kwambiri zokolola zazinthu zazitali za RT-PCR.RNaseH-reverse transcriptase yawonjezeranso kutenthedwa, kotero zomwe zimachitika zimatha kuchitidwa pa kutentha kwambiri kuposa 37-42 ° C.Pansi pa kaphatikizidwe kakulidwe, gwiritsani ntchito oligo(dT) primer ndi 10 μCi ya [α-P]dCTP.Zokolola zonse za strand yoyamba zidawerengedwa pogwiritsa ntchito njira ya mpweya wa TCA.cDNA yautali wonse idawunikidwa pogwiritsa ntchito magulu osanjidwa akulu odulidwa ndikuwerengedwa pa gel wa alkaline agarose.

3. Kwezani kutentha kwa makulitsidwe kuti mulembe mobwerera:

A apamwamba makulitsidwe kutentha kumathandiza kutsegula RNA yachiwiri dongosolo, kuwonjezera zokolola za anachita.Kwa ma templates ambiri a RNA, incubating the RNA ndi primers pa 65 ° C popanda buffer kapena mchere, kutsatiridwa ndi kuzizira kofulumira pa ayezi kudzachotsa mapangidwe ambiri achiwiri ndikulola kuti zoyambazo zimange.Komabe, ma templates ena amakhalabe ndi mawonekedwe achiwiri, ngakhale atatha kutentha.Kukulitsa ma tempuleti ovutawa kutha kuchitidwa pogwiritsa ntchito ThermoScript Reverse Transcriptase ndikuyika zolembera zobwerera m'mbuyo pa kutentha kwakukulu kuti zikulitse bwino.Kutentha kwambiri kwa ma incubation kungathenso kuwonjezereka, makamaka pamene ma gene-specific primers (GSP) amagwiritsidwa ntchito popanga cDNA synthesis (onani Mutu 3).Ngati mukugwiritsa ntchito GSP, onetsetsani kuti Tm ya zoyambira ndizofanana ndi kutentha komwe kumayembekezeredwa.Osagwiritsa ntchito oligo(dT) ndi zoyambira mwachisawawa pamwamba pa 60°C.Zoyambira zosawerengeka zimafuna kukulitsa pa 25°C kwa mphindi 10 zisanachuluke kufika pa 60°C.Kuphatikiza pa kugwiritsa ntchito kutentha kwa reverse reverse transcript, kutsimikizika kungathenso kuwongoleredwa posamutsa mwachindunji kusakaniza kwa RNA/primer kuchoka pa kutentha kwa 65°C kupita ku kutentha kwa reverse transcription incubation ndi kuwonjezera chosakaniza chotenthetsera cha 2×(cDNA hot-start synthesis) .Njirayi imathandiza kupewa kuphatikizika kwa ma intermolecular base komwe kumachitika pa kutentha kochepa.Kusintha kwa kutentha kosiyanasiyana kofunikira pa RT-PCR kumatha kukhala kosavuta pogwiritsa ntchito chozungulira chotenthetsera.

Tth thermostable polymerase imakhala ngati DNA polymerase pamaso pa Mg2+ komanso ngati RNA polymerase pamaso pa Mn2+.Ikhoza kutenthedwa pa kutentha kwakukulu kwa 65 ° C.Komabe, kukhalapo kwa Mn2 + panthawi ya PCR kumachepetsa kukhulupirika, zomwe zimapangitsa Tth polymerase kukhala yosayenerera kukulitsa mwatsatanetsatane, monga cloning ya cDNA.Kuphatikiza apo, Tth imakhala ndi zolembera zocheperako, zomwe zimachepetsa kukhudzidwa, ndipo, popeza kumasulira mobwerezabwereza ndi PCR kumatha kuchitidwa ndi puloteni imodzi, kuwongolera machitidwe popanda kulemberana kumbuyo sikungagwiritsidwe ntchito kufananitsa zinthu zokulitsa za cDNA ndi kuwononga ma genomic DNA.Zogulitsa za amplification zidalekanitsidwa.

4. Zowonjezera zomwe zimalimbikitsa kumasulira mobwerera kumbuyo:

Zowonjezera kuphatikizapo glycerol ndi DMSO zimawonjezeredwa ku njira yoyamba ya kaphatikizidwe, yomwe ingachepetse kukhazikika kwa nucleic acid double-strand ndikumasula dongosolo lachiwiri la RNA.Mpaka 20% glycerol kapena 10% DMSO akhoza kuwonjezeredwa popanda kukhudza SuperScript II kapena MMLV ntchito.AMV imathanso kulekerera mpaka 20% glycerol popanda kutaya ntchito.Kuti muwonjezere kukhudzika kwa RT-PCR mu SuperScriptⅡ reverse transcript reaction, 10% glycerol imatha kuwonjezeredwa ndikuphatikizidwa pa 45°C.Ngati 1/10 ya reverse transcription reaction reaction yawonjezeredwa ku PCR, ndiye kuti kuchuluka kwa glycerol muzochita zokulitsa ndi 0.4%, zomwe sizokwanira kuletsa PCR.

5. Chithandizo cha RNaseH:

Chithandizo cha kaphatikizidwe ka cDNA ndi RNaseH pamaso pa PCR kumatha kukulitsa chidwi.Kwa ma templates ena, amaganiziridwa kuti RNA mu cDNA synthesis reaction imalepheretsa kumangirira kwa zinthu zokulitsa, pomwe chithandizo cha RNaseH chikhoza kukulitsa chidwi.Nthawi zambiri, chithandizo cha RNaseH ndi chofunikira pakukulitsa ma tempuleti a cDNA atalitali, monga otsika-copy tuberous scherosis II.Pachithunzi chovutachi, chithandizo cha RNaseH chinakulitsa chizindikiro chopangidwa ndi SuperScript II kapena cDNA yopangidwa ndi AMV.Pazochitika zambiri za RT-PCR, chithandizo cha RNaseH ndichosankha, chifukwa sitepe ya PCR denaturation pa 95 ° C nthawi zambiri imatulutsa RNA mu RNA: DNA complex.

6. Kupititsa patsogolo Njira Yang'ono Yodziwira za RNA:

RT-PCR imakhala yovuta makamaka ngati pali RNA yochepa chabe.Glycogen yowonjezeredwa ngati chonyamulira panthawi yakudzipatula kwa RNA imathandizira kuchulukitsa zokolola zazing'ono.RNase-free glycogen ikhoza kuwonjezeredwa nthawi yomweyo ndikuwonjezera Trizol.Glycogen imasungunuka m'madzi ndipo imatha kusungidwa mugawo lamadzi ndi RNA kuti ithandizire kugwa kwamvula.Kwa zitsanzo zosakwana 50 mg za minofu kapena maselo 106 otukuka, mlingo woyenera wa RNase-free glycogen ndi 250 μg/ml.

Kuonjezera acetylated BSA ku reverse transcript reaction pogwiritsa ntchito SuperScript II kumatha kukulitsa chidwi, komanso pang'ono RNA, kuchepetsa kuchuluka kwa SuperScript II ndikuwonjezera mayunitsi 40 a RNaseOut nuclease inhibitor kumatha kuwonjezera kuchuluka kwa kuzindikira.Ngati glycogen ikugwiritsidwa ntchito pakudzipatula kwa RNA, tikulimbikitsidwa kuti muwonjezere BSA kapena RNase inhibitor mukamagwiritsa ntchito SuperScript II kuti musinthe kalembedwe.

mwachitsanzo, Wonjezerani RT-PCR mwachindunji

1. CND Asynthesis:

Kaphatikizidwe ka cDNA ka chingwe choyamba chitha kuyambitsidwa pogwiritsa ntchito njira zitatu zosiyana, zomwe zimakhudza kuchuluka ndi mtundu wa cDNA wopangidwa.

Njira yoyambira mwachisawawa inali yocheperako mwa njira zitatuzi.Zoyambira zimayambira pamasamba angapo pazolemba zonse, ndikupanga ma cDNA amfupi, amtali pang'ono.Njirayi imagwiritsidwa ntchito mobwerezabwereza kupeza ma 5 'mapeto otsatizana ndi kupeza cDNA kuchokera ku ma templates a RNA okhala ndi zigawo zachiwiri kapena ndi malo othetsera omwe sangathe kutsatiridwa ndi reverse transcriptase.Kuti mupeze cDNA yayitali kwambiri, chiŵerengero cha zoyambira ku RNA mu RNA iliyonse sampuli ziyenera kutsimikiziridwa mwamphamvu.Kuchuluka koyambira kwa zoyambira mwachisawawa kumachokera ku 50 mpaka 250 ng pa 20 μl zomwe zimachitika.Popeza cDNA yopangidwa kuchokera ku RNA yonse pogwiritsa ntchito zoyambira zosasinthika kwenikweni ndi ribosomal RNA, poly(A) +RNA nthawi zambiri imasankhidwa ngati template.

Oligo (dT) oyambira amakhala achindunji kuposa oyambira mwachisawawa.Imasakanikirana mpaka kumchira wa poly(A) womwe umapezeka kumapeto kwa 3′ ma eukaryotic mRNAs ambiri.Chifukwa poly(A)+ RNA ndi pafupifupi 1% mpaka 2% ya RNA yonse, kuchuluka ndi zovuta za cDNA ndizochepa kwambiri poyerekeza ndi zoyambira mwachisawawa.Chifukwa cha mawonekedwe ake apamwamba, oligo(dT) nthawi zambiri safuna kukhathamiritsa kwa chiŵerengero cha RNA ndi zoyambira ndi poly(A)+ kusankha.Ndibwino kugwiritsa ntchito 0.5μg oligo(dT) pa 20μl reaction system.oligo(dT)12-18 ndi oyenera RT-PCR ambiri.ThermoScript RT-PCR System imapereka oligo(dT)20 chifukwa cha kukhazikika kwake kwa kutentha kwa kutentha kwakukulu kwa ma incubation.

Gene specific primers (GSP) ndizomwe zimayambira kwambiri pamagawo obwereza.GSP ndi oligonucleotide ya antisense yomwe imatha kusakanikirana motsatira ndondomeko ya chandamale ya RNA, mosiyana ndi zoyambira mwachisawawa kapena oligo(dT), zomwe zimayendera ma RNA onse.Malamulo omwewo omwe amagwiritsidwa ntchito popanga zoyambira za PCR amagwiranso ntchito pamapangidwe a GSP mumayendedwe obwereza.GSP ikhoza kukhala yotsatizana ngati choyambira chokulitsa chomwe chimafika kumapeto kwenikweni kwa mRNA, kapena GSP ikhoza kupangidwa kuti ipitirire kumunsi kwa choyambira chokulirapo.Pamitu ina yokulirapo, zoyambira za antisense zimayenera kupangidwira bwino RT-PCR chifukwa mawonekedwe achiwiri a chandamale cha RNA angalepheretse kumangirira koyambira.Ndibwino kugwiritsa ntchito 1 pmol antisense GSP mu 20 μl yoyamba-strand synthesis reaction.

2. Kwezani kutentha kwa makulitsidwe kuti mulembe mobwerera:

Kuti mupindule mokwanira ndi kutsimikizika kwa GSP, reverse transcriptase yokhala ndi thermostability yapamwamba iyenera kugwiritsidwa ntchito.Thermostable reverse transcriptases akhoza kulumikizidwa pa kutentha kwakukulu kuti awonjezere kugunda kwa mtima.Mwachitsanzo, ngati GSP anneals pa 55 ° C, kulunjika kwa GSP sidzagwiritsidwa ntchito mokwanira ngati AMV kapena M-MLV agwiritsidwa ntchito mosintha transcript pa stringency otsika 37°C.Komabe, SuperScript II ndi ThermoScript zitha kuchitapo kanthu pa 50 ° C kapena kupitilira apo, zomwe zimachotsa zinthu zomwe sizili zenizeni zomwe zimapangidwa pakutentha kotsika.Kuti mudziwe zambiri, kusakaniza kwa RNA / primer kumatha kusamutsidwa mwachindunji kuchokera ku 65 ° C kutentha kwa denaturation kupita ku reverse transcript incubation kutentha ndikuwonjezedwa ku 2 × reaction mix mix (cDNA synthesis hot start).Izi zimathandiza kupewa ma intermolecular base pairing pa kutentha kochepa.Kusintha kosiyanasiyana kwa kutentha komwe kumafunikira pa RT-PCR kumatha kukhala kosavuta pogwiritsa ntchito chozungulira chotenthetsera.

3. Amachepetsa kuipitsidwa kwa DNA:

Vuto lomwe lingakhalepo ndi RT-PCR ndikuyipitsidwa kwa genomic DNA mu RNA.Kugwiritsa ntchito njira yabwino yodzipatula ya RNA, monga Trizol Reagent, idzachepetsa kuchuluka kwa DNA ya genomic yomwe imayipitsa kukonzekera kwa RNA.Kupewa zinthu zochokera ku genomic DNA, RNA imatha kuthandizidwa ndi amplification-grade DNase I kuchotsa DNA yoyipitsa isanalembedwenso.Digestion ya DNase I inathetsedwa mwa kuyika zitsanzo mu 2.0 mM EDTA kwa mphindi 10 pa 65 ° C.EDTA imatha kutsitsa ayoni a magnesium, kuteteza RNA hydrolysis yodalira magnesium pa kutentha kwambiri.

Pofuna kulekanitsa cDNA yokulirapo ndi kuipitsa zinthu zokulitsa za DNA, zoyambira zitha kupangidwa kuti chigawo chilichonse chilekanitse ma exons.Zogulitsa za PCR zochokera ku cDNA zidzakhala zazifupi kuposa zomwe zimachokera ku DNA yowonongeka.Kuphatikiza apo, kuyesa koyang'anira popanda kulembedwa m'mbuyo kunkachitika pa template iliyonse ya RNA kuti adziwe ngati chidutswa choperekedwacho chinachokera ku genomic DNA kapena cDNA.Chogulitsa cha PCR chopezedwa popanda kulembedwa m'mbuyo chimachokera ku genome.