Ⅰ. Khulisa ukuzwela kwesistimu yokusabela:

1. Hlukanisa i-RNA yekhwalithi ephezulu:

Ukuhlanganiswa kwe-cDNA okuphumelelayo kuvela ku-RNA yekhwalithi ephezulu.I-RNA yekhwalithi ephezulu kufanele iqinisekise okungenani ingqikithi ende futhi ayiqukethe ama-inhibitor anganawo ama-enzyme okurekhoda, njenge-EDTA noma i-SDS.Izinga le-RNA linquma inani eliphezulu lolwazi lokulandelana ongalulobela ku-cDNA.Indlela evamile yokuhlanza i-RNA iyisinyathelo sesinyathelo sokusebenzisa i-isoocyanate/acidophenol.Ukuze kuvinjelwe ukungcoliswa kwe-RNase, i-RNA ehlukaniswe nesampula ecebile nge-RNase (njengama-pancreas) idinga ukugcinwa kwe-formaldehyde ukuze konge i-RNA yekhwalithi ephezulu, ebaluleke nakakhulu ekugcinweni kwesikhathi eside.I-RNA ekhishwe esibindini samagundane ngokuyisisekelo yonakaliswa ngemva kweviki elilodwa lokugcina emanzini, kuyilapho i-RNA ekhishwe embendeni yamagundane yahlala izinzile ngemva kweminyaka emithathu yokugcina emanzini.Ngaphezu kwalokho, imibhalo emikhulu kuno-4kb izwela kakhulu ukulandelela ukucekelwa phansi kwe-RNase kunemibhalo emincane elotshiwe.Ukuze kwandiswe ukuzinza kwesampula ye-RNA yesitoreji, i-RNA ingancibilika ku-methalmamine ye-ion, futhi igcinwe -70 °C.I-thylide esetshenziselwa ukulondoloza i-RNA akumele iqukathe into exubile eyehlisa isithunzi se-RNA.I-RNA, etholakala kumanyikwe, ingagcinwa ku-methalmamine okungenani unyaka owodwa.Uma usulungele ukusebenzisa i-RNA, ungasebenzisa lezi zindlela ezilandelayo ukuze uthole i-RNA: engeza i-NaCl ku-0.2m futhi izikhathi ezi-4 kumthamo we-ethanol, beka izinga lokushisa legumbi imizuzu engu-3-5, kanye no-10,000 × g centrifugal imizuzu engu-5.

2. Sebenzisa i-reverse transcriptase ngaphandle komsebenzi we-RNaseH (RNaseH-):

Ama-RNase inhibitors avamise ukungezwa ekuphenduleni okulotshiweyo okuhlehliswayo ukuze kwandiswe ubude nesivuno sokuhlanganiswa kwe-cDNA.I-RNase inhibitor yengezwa ekuphenduleni kokuhlanganiswa kweketango lokuqala phambi kwama-buffers nama-ejenti anciphisayo njenge-DTT ngoba inqubo yokuhlanganisa yangaphambi kwe-cDNA ishintsha inhibitor, ngaleyo ndlela ikhulule ama-RNase abophekile alulaza i-RNA.I-protein RNase inhibitor ivimbela kuphela ukuwohloka kwe-RNA nge-RNase A, B, C, futhi ayivimbeli ama-RNase esikhumbeni, ngakho-ke kufanele kuqashelwe ukuthi ungangenisi ama-RNase eminwe 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 uqhudelana nomsebenzi we-polymerase wemicu ye-heterozygous eyakhiwe phakathi kwezifanekiso ze-RNA neziqalo ze-DNA noma imicu yokunweba ye-cDNA, futhi yehlisa isithunzi i-RNA: imicu ye-RNA ku-DNA complex.Izifanekiso ze-RNA ezonakaliswe umsebenzi we-RNaseH ngeke zisakwazi ukusetshenziswa njengama-substrates asebenzayo okuhlanganiswa kwe-cDNA, okunciphisa isivuno nobude bokuhlanganiswa kwe-cDNA.Ngakho ukuqeda noma ukunciphisa kakhulu umsebenzi we-RNaseH we-reverse transcriptase kungaba yinzuzo enkulu.

I-SuperScriptⅡ reverse transcriptase, i-MMLV reverse transcriptase ye-RNaseH- kanye ne-thermoScript reverse transcriptase, i-AMV ye-RNaseH- ikhiqize i-cDNA yobude obugcwele kakhulu kune-MMLV ne-AMV.Ukuzwela kwe-RT-PCR kuthintwa inani le-cDNA ehlanganisiwe.I-ThermoScript izwela kakhulu kune-AMV.Usayizi wemikhiqizo ye-RT-PCR unqunyelwe 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-'s reverse transcriptase iphinde ikhulise ukuzinza kokushisa, ngakho-ke ukusabela kungenziwa emazingeni okushisa aphezulu kunokuvamile okungu-37-42℃.Ngaphansi kwezimo eziphakanyisiwe zokuhlanganisa, ama-primers we-oligo(dT) kanye ne-10μCi [alpha-p]dCTP asetshenzisiwe.Isamba sokukhiqizwa kochungechunge lokuqala kubalwe kusetshenziswa indlela ye-TCA yemvula.I-cDNA yobude obugcwele yahlaziywa kusetshenziswa ukususwa komcu ohlungwe ngosayizi nokubalwa ngejeli ye-alkaline agarose.

3. Khulisa izinga lokushisa lokulondoloza ukushisa lokuloba okuhlanekezelwe:

Ukushisa okungaphezulu kokubamba kusiza ukuvula isakhiwo sesibili se-RNA futhi kwandise isivuno sokusabela.Ezifanekiso eziningi ze-RNA, ukubamba i-RNA kanye ne-primer ku-65°C ngaphandle kwebhafa noma usawoti bese ukuzipholisa ngokushesha eqhweni kuqeda izakhiwo zesibili futhi kuvumela ama-primers ukuthi abophe.Nokho, ezinye izifanekiso zisenesakhiwo sesibili, ngisho nangemva kokushintshashintsha kwe-thermal.Ukukhulisa lezi zifanekiso ezinzima kungenziwa kusetshenziswa i-ThermoScript reverse transcriptase nangokubeka ukusabela kwe-reverse transcriptase emazingeni okushisa aphakeme ukuze kuthuthukiswe ukukhuliswa.Amazinga okushisa aphakeme angakwazi futhi ukwandisa ukucaciswa, ikakhulukazi uma ukuhlanganiswa kwe-cDNA kwenziwa kusetshenziswa izakhi zofuzo ezithize (GSPS) (bona Isahluko 3).Uma usebenzisa i-GSP, qiniseka ukuthi inani le-Tm le-primer liyafana nezinga lokushisa elilindelekile lokubamba.Ungasebenzisi i-oligo(dT) kanye neziqalo ezingahleliwe ezingaphezu kuka-60℃.Ama-primers angahleliwe adinga ukubanjwa ku-25℃ imizuzu eyi-10 ngaphambi kokukhuphuka afike ku-60℃.Ngokungeziwe ekusebenziseni amazinga okushisa aphakeme okulotshiweyo ahlanekezelwe, ukucaciswa kungathuthukiswa ngokudlulisa ngokuqondile ingxube ye-RNA/primer isuka ku-65℃ eshintshayo izinga lokushisa iye kumazinga okushisa okulotshiweyo ahlanekezelwe kanye nokwengeza ingxube yokusabela engu-2× eshisiwe ngaphambili (i-cDNA thermal initiation synthesis).Le ndlela isiza ukuvimbela ukubhanqa kwesisekelo se-intermolecular okwenzeka emazingeni okushisa aphansi.Ukusebenzisa ithuluzi le-PCR kwenza amaswishi amaningi okushisa abe lula adingekayo ku-RT-PCR.

I-Tth ukushisa okuzinzile i-polymerase isebenza njenge-DNA polymerase phambi kwe-Mg2+ ne-RNA polymerase lapho kukhona i-Mn2+.Ingabamba ukushisa kufika ku-65℃.Nokho, ukuba khona kwe-Mn2+ ngesikhathi se-PCR kunciphisa ukwethembeka, okwenza i-Tth polymerase ingakufanelekeli ukukhulisa ukunemba okuphezulu, okufana ne-cDNA cloning.Ukwengeza, i-Tth ayisebenzi kahle ekubhalweni okuhlanekezelwe, okunciphisa ukuzwela, futhi njengoba i-enzyme eyodwa ingakwazi ukwenza ukuloba okuhlanekezelwe kanye ne-PCR, ukulawula ukusabela ngaphandle kokuloba okuhlanekezelwe ngeke kusetshenziselwe ukuhlukanisa imikhiqizo ekhulisiwe ye-cDNA kuleyo ye-DNA ye-genomic engcolile.

4. Isengezo esithuthukisa ukuloba okuhlanekezelwe:

Ukwengezwa kwezithasiselo, okuhlanganisa i-glycerin ne-DMSO, ekuphenduleni kokuqala kwe-chain synthesis kunganciphisa ukuzinza kwe-nucleic acid double strand futhi kukhulule isakhiwo sesibili se-RNA.Kungafika ku-20% we-glycerin noma u-10% we-DMSO ngaphandle kokuthikameze umsebenzi we-SuperScriptⅡ noma we-MMLV.I-AMV ingakwazi futhi ukubekezelela kufika ku-20% we-glycerol ngaphandle kokunciphisa umsebenzi.Ukuze ukhulise ukuzwela kwe-RT-PCR ku-SuperScriptⅡ reverse transcription reaction, u-10% we-glycerol ungangezwa futhi ugqunywe ku-45℃.Uma i-1/10 yomkhiqizo we-retrotranscription-reaction yengezwa ku-PCR, ukugxila kwe-glycerol ekuphenduleni kokukhulisa kungu-0.4%, okungenele ukuvimbela i-PCR.

5. Ukucubungula kwe-RNaseH:

Ukuzwela kungathuthukiswa ngokwelapha ukusabela kokuhlanganiswa kwe-cDNA nge-RNaseH ngaphambi kwe-PCR.Kwezinye izifanekiso, kucatshangwa ukuthi i-RNA ku-cDNA synthesis reaction ivimbela ukuboshwa kwemikhiqizo ekhulisiwe, lapho ukwelapha kwe-RNaseH kungakhuphula ukuzwela.Ngokuvamile, ukwelashwa kwe-RNaseH kuyadingeka ukuze kukhuliswe ithempulethi eqondiwe ye-cDNA enobude obugcwele, njenge-tuberous scheresisⅡ enekhophi ephansi.Kulesi sifanekiso esinzima, i-RNaseH ithuthukise isignali ekhiqizwe i-cDNA ehlanganiswe yi-SuperScriptⅡ noma i-AMV.Ngokusabela okuningi kwe-RT-PCR, ukwelashwa kwe-RNaseH kungokuzithandela ngoba isinyathelo sokuguqula i-PCR esivalekile esingu-95℃ sivamise ukuguqula i-RNA isuke ku-RNA: DNA complex.

6. Izindlela ezithuthukisiwe zokuthola amanani amancane e-RNA:

I-RT-PCR iyinselele ikakhulukazi uma amanani amancane e-RNA atholakalayo.Ukwengezwa kwe-glycogen njengesithwali ngesikhathi sokuhlukaniswa kwe-RNA kusiza ukwandisa isivuno samasampula amancane.I-glycogen engenayo i-RNase ingangezwa ngesikhathi esifanayo ne-Trizol.I-Glycogen iyancibilika emanzini futhi ingahlala esigabeni samanzi ne-RNA ukuze isize emvuleni elandelayo.Ukuhlushwa okunconyiwe kwe-RNase-free glycogen ngu-250μg/ml kumasampuli angaphansi kuka-50mg wezicubu noma amaseli akhulisiwe angu-106.

Ukwengezwa kwe-acetylated BSA ukubuyisela emuva ukusabela kokulotshiweyo kusetshenziswa i-SuperScriptⅡ kungakhuphula ukuzwela, futhi ngamanani amancane e-RNA, ukwehlisa inani le-SuperScriptⅡ nokwengeza amayunithi angu-40 e-RnaseOut nuclease inhibitor kungathuthukisa izinga lokutholwa.Uma i-glycogen isetshenziswa ekuhlukaniseni kwe-RNA, ukungezwa kwe-BSA noma i-RNase inhibitors ukubuyisela emuva ukusabela kokulotshiweyo kusetshenziswa i-SuperScriptⅡ kusanconywa.

Ⅱ. Khulisa ukucaciswa kwe-RT-PCR

1. Ukuhlanganiswa kwe-cNDA:

Izindlela ezintathu ezihlukene zingasetshenziswa ukuqalisa ukuhlanganiswa kwe-cDNA yomucu wokuqala, futhi ukucaciswa okuhlobene kwendlela ngayinye kuthinta inani nohlobo lwe-cDNA ehlanganisiwe.

Indlela ye-primer engahleliwe iyona ndlela eqondile kakhulu kulezi zindlela ezintathu.Ama-Primers abanjwa kumasayithi amaningi kuwo wonke umbhalo ukuze kukhiqizwe i-cDNA efushane, ubude obuyingxenye.Le ndlela ivame ukusetshenziswa ukuze kutholwe 5′ amatheminali alandelanayo kanye ne-cDNA ezifanekisweni ze-RNA ezinezifunda zesakhiwo sesibili noma nezingosi zokunqamula i-reverse transcriptase engakwazi ukuphindaphinda.Ukuze kutholwe i-cDNA ende kakhulu, isilinganiso sama-primers ukuya ku-RNA kusampula ngayinye ye-RNA sidinga ukunqunywa ngokwamandla.Ukugxila kokuqala kwama-primers okungahleliwe kusuka ku-50 kuye ku-250ng ngesistimu yokusabela engu-20μl.Ngenxa yokuthi i-cDNA ehlanganiswe ku-RNA iyonke kusetshenziswa ama-primers angahleliwe ngokuyinhloko i-ribosomal RNA, i-poly(A)+RNA ngokuvamile ikhethwa njengesifanekiso.

Ukuqaliswa kwe-Oligo(dT) kucace kakhulu kuneziqalo ezingahleliwe.Ihlanganiswa nomsila we-poly(A) otholakala ekupheleni kuka-3′ we-mRNA kumaseli amaningi e-eukaryotic.Ngenxa yokuthi i-poly(A)+RNA icishe ibe ngu-1% kuya ku-2% wesamba se-RNA, inani nobunkimbinkimbi be-cDNA buncane kakhulu uma kusetshenziswe iziqalo ezingahleliwe.Ngenxa yokucaciswa kwayo okuphezulu, i-oligo(dT) ngokuvamile ayidingi ukulungiselelwa kwe-RNA kuya kusilinganiso sokuqala 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 ihlinzeka nge-oligo(dT)20 ngenxa yokuqina kwayo okuhle kwe-thermal futhi ifanele amazinga okushisa okubamba aphezulu.

Ama-primers we-Gene-specific (GSP) ayiziqalisi eziqondile ezingcono kakhulu zesinyathelo sokuhlehla sokuloba.I-GSP iyi-oligonucleoside ephikisayo engakwazi ukuhlanganisa ngokuqondile ngokulandelana kwendawo okuyiwa kuyo ye-RNA, esikhundleni sokuhlanganisa wonke ama-Rna afana nama-random primers noma i-oligo(dT).Imithetho esetshenziselwa ukuklama ama-primers e-PCR futhi iyasebenza ekwakhiweni kokusabela kokulotshiweyo okuhlanekezelwe i-GSP.I-GSP ingaba ukulandelana okufanayo njengesiqalo sokukhulisa i-amplification esifakwe ekugcineni kwe-mRNA3′, noma i-GSP ingaklanywa ukuthi idonswe phansi ngomfula nge-primer yokukhulisa ehlehlayo.Kwezinye izinto ezikhulisiwe, kuyadingeka ukuba udizayine i-primer ye-antisense engaphezu kweyodwa ye-RT-PCR ephumelelayo ngoba ukwakheka kwesibili kwe-RNA eqondiwe kungase kuvimbele i-primer ekubopheni.Kuphakanyiswa ukuthi kusetshenziswe i-1pmol antisense GSP ohlelweni lokuqala lwe-chain synthesis reaction lwe-20μl.

2. Khulisa izinga lokushisa lokulondoloza ukushisa lokuloba okuhlanekezelwe:

Ukuze usebenzise ngokugcwele ukucaciswa kwe-GSP, i-reverse transcriptase enokuqina kokushisa okuphezulu kufanele isetshenziswe.I-heat-stable reverse transcriptase ingafakwa emazingeni okushisa aphezulu ukuze kwandiswe ukusabela okuqinile.Isibonelo, uma i-GSP ixhunywe ku-55°C, khona-ke ukucaciswa kwe-GSP akusetshenziswa ngokugcwele uma ukuloba okuhlanekezelwe kwenziwa ngo-37°C ngokuqina okuphansi kusetshenziswa i-AMV noma i-M-MLV.Nokho, i-SuperScripⅡ ne-ThermoScript zingasabela kokuthi 50℃ noma ngaphezulu, okuqeda imikhiqizo engaqondile ekhiqizwa emazingeni okushisa aphansi.Ukuze uthole ukucaciswa okuphezulu, ingxube ye-RNA/primer ingadluliswa ngokuqondile ukusuka ku-65℃ izinga lokushisa lokuguqula i-denaturation iye emazingeni okushisa aphethe okulotshiweyo okuhlanekezelwe ngokungezwa kwengxube yokusabela engu-2 x eshisiwe ngaphambili (ukuqaliswa okushisayo kwe-cDNA synthesis).Lokhu kusiza ukuvimbela ukubhanqa kwesisekelo phakathi kwama-molecule emazingeni okushisa aphansi.Ukusebenzisa ithuluzi le-PCR kwenza kube lula izinguquko eziningi zokushisa ezidingekayo ku-RT-PCR.

3. Yehlisa ukungcoliswa kwe-DNA ye-genomic:

Obunye ubunzima obungaba khona nge-RT-PCR ukuthi i-RNA ingcolisa i-genomic DNA.Ukusetshenziswa kwezindlela ezingcono zokuhlukanisa i-RNA, njenge-Trizol Reagent, kunciphisa ukungcoliswa kwe-DNA ye-genomic kumalungiselelo e-RNA.Ukuze ugweme imikhiqizo ekhiqizwe ku-DNA ye-genomic, i-RNA ingelashwa nge-amplification grade DnasⅠ ukuze kukhishwe i-DNA engcolile ngaphambi kokuhlehlisa ukulotshwa.Amasampuli agcinwe ku-65℃ ku-2.0mM EDTA imizuzu engu-10 ukuze kuqedwe ukugaya kwe-DNaseⅠ.I-EDTA ichelela ama-ion e-magnesium ukuvimbela i-RNA hydrolysis encike ku-magnesium ion eyenzeka emazingeni okushisa aphezulu.

Ukuze kuhlukaniswe i-cDNA ekhulisiwe kumkhiqizo wokukhulisa i-DNA we-genome, ama-primer ahlanganiswa ngokuhlukile nge-exon ehlukanisiwe angaklanywa.Imikhiqizo ye-PCR ethathwe ku-cDNA izoba mifishane kunaleyo etholakala ku-DNA ye-genomic engcolile.Ukuhlolwa okulawulwayo ngaphandle kokulotshwa okuhlanekezelwe nakho kwenziwa kusifanekiso ngasinye se-RNA ukuze kunqunywe ukuthi ucezu olunikeziwe lusuka ku-genomic DNA noma i-cDNA.Imikhiqizo ye-PCR etholwe ngaphandle kokulotshwa okuhlanekezelwe isuselwa ku-genome.

Umkhiqizo Ohlobene

I-RT-PCR Kulula^ᵀᴹI(Isinyathelo esisodwa)

-Ikhithi yesinyathelo esisodwa yenza ukuloba okuhlanekezelwe kanye ne-PCR kwenziwe ngeshubhu efanayo.Idinga kuphela ukwengeza isifanekiso se-RNA, iziqalisi ze-PCR ezithile kanye ne-ddH Yamahhala ye-RNase₂O.

-Real-time Ukuhlaziywa kobuningi be-RNA kungenziwa ngokushesha nangokunembile.

-Ikhithi isebenzisa i-Foregene reverse transcription reagent eyingqayizivele kanye ne-Foregene HotStar Taq DNA Polymerase ehlanganiswe nesistimu yokusabela eyingqayizivele ukuze kuthuthukiswe ngempumelelo ukukhulisa ubuciko kanye nokucaciswa kokusabela.

-Isistimu yokusabela eyenziwe kahle yenza ukusabela kube nokuzwela okuphezulu kokutholwa, ukuqina okushisayo okuqinile, nokubekezelelana okungcono.

I-RT Easy II(Nge-GDNase) I-Master Premix Ye-First-Strand CDNA Synthesis Yesikhathi Sangempela PCR Nge-GDNase

-Ikhono elisebenzayo lokususa i-gDNA, engasusa i-gDNA kusifanekiso phakathi nemizuzu emi-2.

-Isistimu yokubhala ehlehlayo esebenza kahle, kuthatha imizuzu eyi-15 kuphela ukuqeda ukuhlanganiswa kwe-cDNA yomucu wokuqala.

-Izifanekiso eziyinkimbinkimbi: izifanekiso ezinokuqukethwe okuphezulu kwe-GC nesakhiwo sesibili esiyinkimbinkimbi nazo zingahlehliswa ngokusebenza kahle okuphezulu.

-I-High-sensitivity reverse transcription system, izifanekiso zezinga le-pg nazo zingathola i-cDNA yekhwalithi ephezulu.

-Isistimu yokubhala ngokuhlehlayo inokuqina okuphezulu kwe-thermal, izinga lokushisa elilungile lokusabela ngu-42℃, futhi isenokusebenza okuhle kokulotshiweyo okuhlehlayo kokuthi 50℃.