一、 Khulisa ukuzwela kwesistimu yokusabela:

1. Hlukanisa i-RNA yekhwalithi ephezulu:

Ukuhlanganiswa kwe-cDNA okuphumelelayo kuvela ku-RNA yekhwalithi ephezulu.I-RNA yekhwalithi ephezulu kufanele okungenani ibe ubude obugcwele futhi ingabi nama-reverse transcriptase inhibitors njenge-EDTA noma i-SDS.Izinga le-RNA linquma inani eliphezulu lolwazi lokulandelana ongalulobela ku-cDNA.Indlela evamile yokuhlanza i-RNA yindlela enesinyathelo esisodwa esebenzisa i-guanidine isothiocyanate/i-acid phenol.Ukuze uvimbele ukungcoliswa ngamanani okulandelela e-RNase, i-RNA ehlukanisiwe kumasampula anothe nge-RNase (njengama-pancreas) idinga ukugcinwa ku-formaldehyde ukuze kulondolozwe i-RNA yekhwalithi ephezulu, ikakhulukazi ukuze igcinwe isikhathi eside.I-RNA ekhishwe esibindini samagundane yonakaliswa ngokuyisisekelo ngemva kokugcinwa emanzini isonto elilodwa, kuyilapho i-RNA ekhishwe ubende lwamagundane yahlala izinzile ngemva kokugcinwa emanzini iminyaka engu-3.Ngokwengeziwe, okulotshiweyo okude kuno-4 kb kuzwela kakhulu ekwehlisweni ngama-trace RNases kunemibhalo emincane.Ukuze kwandiswe ukuzinza kwamasampula e-RNA agciniwe, i-RNA ingancibilika ku-formamide eyenziwe nge-deionized futhi igcinwe ku -70°C.I-Formamide esetshenziselwa ukulondoloza i-RNA kufanele ingabi nemfucumfucu eyehlisa isithunzi ye-RNA.I-RNA evela kumanyikwe ingagcinwa ku-formamide okungenani unyaka owodwa.Uma ulungiselela ukusebenzisa i-RNA, ungasebenzisa le ndlela elandelayo ukuze uthole i-RNA: engeza i-NaCl ku-0.2M futhi izikhathi ezi-4 ivolumu ye-ethanol, beka ekamelweni lokushisa imizuzu engu-3-5, kanye ne-centrifuge ku-10,000 × g imizuzu engu-5.

2. Sebenzisa i-RNaseH-inactive (RNaseH-) reverse transcriptase:

Ama-RNase inhibitors avamise ukungezwa ekuphenduleni okulotshiweyo okuhlehliswayo ukuze kwandiswe ubude nesivuno sokuhlanganiswa kwe-cDNA.I-RNase inhibitors kufanele yengezwe ngesikhathi sokusabela kokuhlanganiswa komucu wokuqala phambi kwe-bafa kanye ne-ejenti yokunciphisa (njenge-DTT), ngoba inqubo ngaphambi kokuhlanganiswa kwe-cDNA isusa inhibitor, ngaleyo ndlela ikhulule i-RNase eboshiwe engehlisa isithunzi i-RNA.Ama-protein RNase inhibitors avimbela kuphela ukuwohloka kwe-RNA nge-RNase A, B, C, futhi awavimbeli i-RNase esikhumbeni, ngakho-ke qaphela ukuthi ungangenisi i-RNase eminweni yakho naphezu kokusetshenziswa kwalezi zivimbela.

I-Reverse transcriptase yenza ukuguqulwa kwe-RNA ibe yi-cDNA.Kokubili i-M-MLV ne-AMV inomsebenzi we-RNaseH ongapheli ngaphezu komsebenzi wayo we-polymerase.Umsebenzi we-RNaseH nomsebenzi we-polymerase uqhudelana wodwa nge-hybrid strand eyakhiwe phakathi kwesifanekiso se-RNA kanye ne-DNA primer noma i-cDNA extension strand, futhi yehlisa isithunzi i-RNA strand ku-RNA:DNA complex.Isifanekiso se-RNA esehliswe umsebenzi we-RNaseH ngeke sisakwazi ukusebenza njenge-substrate esebenzayo yokuhlanganiswa kwe-cDNA, enciphisa isivuno nobude bokuhlanganiswa kwe-cDNA.Ngakho-ke, kungaba yinzuzo ukuqeda noma ukunciphisa kakhulu umsebenzi we-RNaseH we-reverse transcriptase..

I-SuperScript Ⅱ i-reverse transcriptase, i-RNaseH- MMLV reverse transcriptase ne-thermoScript reverse transcriptase, i-RNaseH- AMV, ingathola inani eliningi nobude obugcwele be-cDNA kune-MMLV ne-AMV.Ukuzwela kwe-RT-PCR kuzothintwa inani le-cDNA synthesis.I-ThermoScript izwela kakhulu kune-AMV.Usayizi wemikhiqizo ye-RT-PCR ukhawulelwe ikhono le-reverse transcriptase ukuze kuhlanganiswe i-cDNA, ikakhulukazi uma kuhlanganisa ama-cDNA amakhulu.Uma kuqhathaniswa ne-MMLV, i-SuperScripⅡ ikhulise kakhulu umkhiqizo wemikhiqizo ende ye-RT-PCR.I-RNaseH-reverse transcriptase nayo inyuse izinga lokushisa, ngakho ukusabela kungenziwa emazingeni okushisa angaphezu kuka-37-42°C evamile.Ngaphansi kwezimo eziphakanyisiwe zokwenziwa, sebenzisa i-oligo(dT) primer kanye no-10 μCi we-[α-P]dCTP.Isamba sesivuno somucu wokuqala sibalwa kusetshenziswa indlela ye-TCA yemvula.I-cDNA yobude obugcwele yahlaziywa kusetshenziswa amabhendi ahlungwe ngosayizi anqanyuliwe futhi abalwa kujeli ye-alkaline agarose.

3. Phakamisa izinga lokushisa ekufukameleni ukuze ulotshwe ngokuhlehla:

Ukushisa okuphezulu kwe-incubation kusiza ukuvula isakhiwo sesibili se-RNA, okwandisa isivuno sokusabela.Ezifanekiso eziningi ze-RNA, ukufukamela i-RNA nama-primers ku-65°C ngaphandle kwebhafa noma usawoti, okulandelwa ukupholisa ngokushesha eqhweni kuzoqeda izakhiwo eziningi zesibili futhi kuvumele ama-primers ukuthi abophe.Kodwa-ke, ezinye izifanekiso zisenezakhiwo zesibili, ngisho nangemva kokukhishwa kwe-denaturation.Ukukhulisa lezi zifanekiso ezinzima kungenziwa kusetshenziswa i-ThermoScript Reverse Transcriptase futhi kubekwe ukusabela okuhlanekezelwe kokulotshiweyo ezingeni lokushisa eliphezulu ukuze kuthuthukiswe ukukhula.Amazinga okushisa aphakeme e-incubation nawo angakhuphula ukucaciswa, ikakhulukazi uma ama-primers e-gene-specific (GSP) asetshenziselwa ukuhlanganiswa kwe-cDNA (bona Isahluko 3).Uma usebenzisa i-GSP, qinisekisa ukuthi i-Tm yama-primers iyafana nezinga lokushisa elilindelekile lokufukamela.Ungasebenzisi i-oligo(dT) kanye neziqalo ezingahleliwe ezingaphezu kuka-60°C.Ama-primers angahleliwe adinga ukufukamela ku-25°C imizuzu eyi-10 ngaphambi kokukhuphuka afike ku-60°C.Ngokungeziwe ekusebenziseni izinga lokushisa eliphezulu lokuloba okuhlanekezelwe, ukucaciswa kungathuthukiswa ngokudlulisa ngokuqondile imiksi ye-RNA/primer isuka ku-65°C yokushintshashintsha kwezinga lokushisa iye emazingeni okushisa ahlanekezelwe e-transcription incubation kanye nokwengeza ingxube yokusabela engu-2× efudumele ngaphambili (i-cDNA hot-start synthesis) .Le ndlela isiza ukuvimbela ukubhanqa kwesisekelo se-intermolecular okwenzeka emazingeni okushisa aphansi.Ukushintsha kwezinga lokushisa okuningana okudingekayo ku-RT-PCR kungenziwa lula ngokusebenzisa umjikelezo oshisayo.

I-polymerase ye-Tth thermostable isebenza njenge-DNA polymerase phambi kwe-Mg2+ nanjenge-RNA polymerase phambi kwe-Mn2+.Ingagcinwa ifudumele ekushiseni okuphezulu okungama-65°C.Kodwa-ke, ukuba khona kwe-Mn2+ ngesikhathi se-PCR kunciphisa ukwethembeka, okwenza i-Tth polymerase ingakufanelekeli ukukhulisa ukunemba okuphezulu, njengokuhlanganisa i-cDNA.Ukwengeza, i-Tth ine-reverse transcription ephansi esebenza kahle, enciphisa ukuzwela, futhi, njengoba ukuloba okuhlanekezelwe kanye ne-PCR kungenziwa nge-enzyme eyodwa, ukusabela kokulawula ngaphandle kokuloba okuhlanekezelwe ngeke kusetshenziselwe ukuqhathanisa imikhiqizo yokukhulisa i-cDNA ne-DNA engcolisayo ye-genomic.Imikhiqizo yokukhulisa i-amplification yahlukaniswa.

4. Izengezo ezikhuthaza ukuloba okuhlanekezelwe:

Izithasiselo ezihlanganisa i-glycerol ne-DMSO zengezwa ekuphenduleni kokuqala kwe-strand synthesis, okunganciphisa ukuzinza kwe-nucleic acid kabili-strand futhi kukhulule isakhiwo sesibili se-RNA.Kungafika ku-20% we-glycerol noma u-10% we-DMSO angangezwa ngaphandle kokuthikameza umsebenzi we-SuperScript II noma we-MMLV.I-AMV ingakwazi futhi ukubekezelela kufika ku-20% we-glycerol ngaphandle kokulahlekelwa umsebenzi.Ukuze kwandiswe ukuzwela kwe-RT-PCR ku-SuperScriptⅡ reverse transcription reaction, 10% glycerol ingangezwa futhi ifakwe ku-45°C.Uma i-1/10 yomkhiqizo wokusabela okulotshiwe okuhlanekezelwe ingezwa ku-PCR, khona-ke ukugcwala kwe-glycerol ekuphenduleni kokukhulisa kungu-0.4%, okungenele ukuvimbela i-PCR.

5. Ukwelashwa kwe-RNaseH:

Ukwelashwa kokusabela kokuhlanganiswa kwe-cDNA nge-RNaseH ngaphambi kwe-PCR kungakhuphula ukuzwela.Kwezinye izifanekiso, kucatshangwa ukuthi i-RNA ekuphenduleni kokuhlanganiswa kwe-cDNA ivimbela ukuboshwa kwemikhiqizo yokukhulisa izwi, lapho ukwelashwa kwe-RNaseH kungandisa ukuzwela.Ngokuvamile, ukwelashwa kwe-RNaseH kuyadingeka lapho kukhuliswa izifanekiso eziqondiwe ze-cDNA ezinobude obugcwele, njengekhophi ephansi ye-tuberous scherose II.Kulesi sifanekiso esinzima, ukwelashwa kwe-RNaseH kuthuthukise isignali ekhiqizwe i-SuperScript II noma i-cDNA ehlanganiswe ne-AMV.Ngokusabela okuningi kwe-RT-PCR, ukwelashwa kwe-RNaseH kungokuzithandela, ngoba isinyathelo sokuguqula isimo se-PCR kokuthi 95°C sivamise ukwenza i-RNA i-hydrolyze ku-RNA:DNA complex.

6. Ukuthuthukiswa Kwendlela Yokuthola I-RNA Encane:

I-RT-PCR iyinselele ikakhulukazi uma amanani amancane e-RNA atholakalayo.I-Glycogen engezwe njengesithwali ngesikhathi sokuhlukaniswa kwe-RNA isiza ukukhulisa isivuno samasampula amancane.I-glycogen engenayo i-RNase ingangezwa ngesikhathi esifanayo nokwengeza i-Trizol.I-Glycogen iyancibilika emanzini futhi ingagcinwa esigabeni esimanzi nge-RNA ukusiza ukuna okulandelayo.Kumasampuli angaphansi kuka-50 mg wezicubu noma amaseli akhulisiwe angu-106, ukugxiliswa okunconyiwe kwe-RNase-free glycogen ngu-250 μg/ml.

Ukwengeza i-acetylated BSA ekuphenduleni kokulotshiweyo okuhlanekezelwe kusetshenziswa i-SuperScript II kungakhuphula ukuzwela, futhi ngamanani amancane e-RNA, ukwehlisa inani le-SuperScript II nokwengeza amayunithi angu-40 e-RNaseOut nuclease inhibitor kungakhuphula izinga lokutholwa.Uma i-glycogen isetshenziswa enqubweni yokuhlukanisa i-RNA, kusanconywa ukuthi wengeze i-BSA noma i-RNase inhibitor lapho usebenzisa i-SuperScript II yokusabela kokuhlehla kokulotshiweyo.

, Nyusa ukucaciswa kwe-RT-PCR

1. I-CND Asynthesis:

Ukuhlanganiswa kwe-cDNA yomucu wokuqala kungaqalwa kusetshenziswa izindlela ezintathu ezihlukene, ukucaciswa okuhlobene okuthinta inani nohlobo lwe-cDNA ehlanganisiwe.

Indlela ye-primer engahleliwe yayingaqondile kakhulu kulezi zindlela ezintathu.Ama-primer atholakala kumasayithi amaningi kuwo wonke umbhalo, akhiqiza ama-cDNA amafushane, anobude obuyingxenye.Le ndlela isetshenziswa kaningi ukuze kutholwe ukulandelana kokuphela okungu-5 nokuthola i-cDNA ezifanekisweni ze-RNA ezinezifunda zesakhiwo sesibili noma ngezingosi zokuqeda ezingakwazi ukuphindwa yi-reverse transcriptase.Ukuze kutholwe i-cDNA ende kakhulu, isilinganiso sama-primers ukuya ku-RNA kusampula ngayinye ye-RNA sidinga ukunqunywa ngokwamandla.Ukugxila kokuqala kwama-primers okungahleliwe kwakusukela ku-50 kuye ku-250 ng ku-20 μl yokusabela.Njengoba i-cDNA ihlanganiswe kusuka ku-RNA iyonke kusetshenziswa iziqalo ezingahleliwe ngokuyinhloko i-ribosomal RNA, i-poly(A)+RNA ngokuvamile ikhethwa njengesifanekiso.

Iziqalo ze-Oligo(dT) zicace kakhulu kuneziqalo ezingahleliwe.Ihlanganisa umsila we-poly(A) otholakala ekugcineni okungu-3′ kwamaningi e-eukaryotic mRNAs.Ngenxa yokuthi i-poly(A)+ RNA icishe ibe ngu-1% kuya ku-2% wesamba se-RNA, inani nobunkimbinkimbi be-cDNA buncane kakhulu kuneziqalo ezingahleliwe.Ngenxa yokucaciswa kwayo okuphezulu, i-oligo(dT) ngokuvamile ayidingi ukuthuthukiswa kwesilinganiso se-RNA kuma-primers kanye nokukhetha kwe-poly(A)+.Kunconywa ukusebenzisa i-0.5μg oligo(dT) ngesistimu yokusabela engu-20μl.i-oligo(dT)12-18 ifanele i-RT-PCR eminingi.I-ThermoScript RT-PCR System inikezela nge-oligo(dT)20 ngenxa yokuqina kwayo okushisayo kwamazinga okushisa aphezulu e-incubation.

I-Gene specific primers (GSP) yiziqalo eziqondile kakhulu zesinyathelo sokuhlehla sokuloba.I-GSP iyi-oligonucleotide ephikisayo engakwazi ukuhlanganisa ngokuqondile ngokulandelana kwethagethi ye-RNA, ngokungafani nama-primers angahleliwe noma i-oligo(dT), ehlanganisa wonke ama-RNA.Imithetho efanayo esetshenziselwa ukuklama ama-primers e-PCR iyasebenza ekwakhiweni kwe-GSP ekuphenduleni kokuhlehla kokulotshiweyo.I-GSP ingaba ukulandelana okufanayo njenge-primer yokukhulisa efinyelela ekugcineni okungu-3′-iningi le-mRNA, noma i-GSP ingadizayinelwa ukuhlanganisa ezansi nomfula we-primer yokukhulisa u-reverse.Kwezinye izifundo ezikhulisiwe, i-antisense primer engaphezu kweyodwa idinga ukudizayinelwa i-RT-PCR eyimpumelelo ngoba ukwakheka kwesibili kwe-RNA eqondiwe kungase kuvimbele ukubophezela kwe-primer.Kunconywa ukusebenzisa i-1 pmol antisense GSP ku-20 μl yokusabela kokuhlanganiswa komucu wokuqala.

2. Phakamisa izinga lokushisa ekufukameleni ukuze ubhale ngokuhlehla:

Ukuze usebenzise ngokugcwele inzuzo egcwele yokucaciswa kwe-GSP, i-reverse transcriptase ene-thermostability ephakeme kufanele isetshenziswe.I-Thermostable reverse transcriptases ingafukanyelwa emazingeni okushisa aphezulu ukuze kwandiswe ukuqina kokusabela.Isibonelo, uma i-GSP ifinyelela ku-55°C, ukucaciswa kwe-GSP ngeke kusetshenziswe ngokugcwele uma i-AMV noma i-M-MLV isetshenziselwa ukuloba okuhlanekezelwe nge-stringency ephansi engu-37°C.Nokho, i-SuperScript II ne-ThermoScript zingaphendulwa ku-50°C noma ngaphezulu, okuzosusa imikhiqizo engaqondile ekhiqizwa emazingeni okushisa aphansi.Ukuze uthole ukucaciswa okuphezulu, i-RNA/primer mix ingadluliswa ngokuqondile isuka ku-65°C izinga lokushisa lokushintshashintshashintshashintshayo iye emazingeni okushisa ahlanekezelwe e-reverse transcription futhi yengezwe kungxube yokusabela engu-2× efudunyeziwe ngaphambili (isiqalo esishisayo se-cDNA).Lokhu kusiza ukuvimbela ukubhanqa kwesisekelo se-intermolecular emazingeni okushisa aphansi.Ukushintsha kwezinga lokushisa okuningana okudingekayo ku-RT-PCR kungenziwa lula ngokusebenzisa isithuthukisi esishisayo.

3. Yehlisa ukungcoliswa kwe-DNA ye-genomic:

Ubunzima obungaba khona nge-RT-PCR ukungcoliswa kwe-genomic DNA ku-RNA.Ukusebenzisa indlela enhle yokuhlukanisa i-RNA, njenge-Trizol Reagent, kuzonciphisa inani le-genomic DNA engcolisa ukulungiswa kwe-RNA.Ukuze ugweme imikhiqizo ethathwe ku-DNA ye-genomic, i-RNA ingelashwa nge-amplification-grade DNase I ukuze kususwe i-DNA engcolisayo ngaphambi kokuhlehlisa ukuloba.Ukugaya kwe-DNase I kuqedwe ngokufukamela amasampula ku-2.0 mM EDTA imizuzu eyi-10 ku-65°C.I-EDTA ingakwazi ukugaya i-magnesium ion, ivimbele i-RNA hydrolysis encike ku-magnesium ion emazingeni okushisa aphezulu.

Ukuze kuhlukaniswe i-cDNA ekhulisiwe ekungcoliseni imikhiqizo yokukhulisa i-DNA ye-genomic, ama-primers angaklanywa ukuthi i-anneal ngayinye ihlukanise ama-exons.Imikhiqizo ye-PCR ethathwe ku-cDNA izoba mifishane kunaleyo etholakala ku-DNA ye-genomic engcolile.Ngaphezu kwalokho, ukuhlolwa kokulawula ngaphandle kokulotshwa okuhlanekezelwe kwenziwa kusifanekiso ngasinye se-RNA ukuze kunqunywe ukuthi ucezu olunikeziwe luthathwe ku-genomic DNA noma i-cDNA.Umkhiqizo we-PCR otholwe ngaphandle kokulotshwa okuhlanekezelwe ususelwa ku-genome.