一、 Eketsa kutloisiso ea sistimi ea karabelo:

1. Arola RNA ea boleng bo holimo:

CDNA synthesis e atlehileng e tsoa ho RNA ea boleng bo holimo.RNA ea boleng bo holimo bonyane e lokela ho ba bolelele bo felletseng 'me e se na li-reverse transcriptase inhibitors tse kang EDTA kapa SDS.Boleng ba RNA bo etsa qeto ea palo e kholo ea tlhaiso-leseling eo u ka e ngollang ho cDNA.Mokhoa o tloaelehileng oa ho hloekisa RNA ke mokhoa oa mohato o le mong o sebelisang guanidine isothiocyanate / acid phenol.Ho thibela tšilafalo ka ho latela palo ea RNase, RNA e arohaneng le lisampole tse ruileng tsa RNase (joalo ka manyeme) e hloka ho bolokoa ka formaldehyde ho boloka RNA ea boleng bo holimo, haholo bakeng sa polokelo ea nako e telele.RNA e ntšitsoeng sebeteng sa rat e ile ea senyeha ka mor'a ho bolokoa ka metsing beke e le 'ngoe, ha RNA e ntšitsoeng ho rat spleen e ile ea lula e tsitsitse ka mor'a ho bolokoa ka metsing ka lilemo tse 3.Ho feta moo, lingoloa tse telele ho feta 4 kb li na le kutloelo-bohloko haholo ho theoloeng ke trace RNases ho feta lingoliloeng tse nyane.Ho eketsa botsitso ba disampole tsa RNA tse bolokilweng, RNA e ka qhibidiha ka deionized formamide mme ya bolokwa ho -70°C.Formamide e sebelisetsoang ho boloka RNA e tlameha ho hloka litšila tse senyang RNA.RNA e tsoang manyeme e ka bolokoa ka formamide bonyane selemo.Ha u itokisetsa ho sebelisa RNA, u ka sebelisa mokhoa o latelang ho fokotsa RNA: eketsa NaCl ho 0.2M le makhetlo a 4 ho feta molumo oa ethanol, sebaka sa mocheso oa kamore bakeng sa metsotso e 3-5, le centrifuge ka 10,000 × g metsotso e 5.

2. Sebelisa RNaseH-inactive (RNaseH-) reverse transcriptase:

RNase inhibitors hangata e eketsoa ho fetola karabelo ea transcript ho eketsa bolelele le chai ea motsoako oa cDNA.Li-inhibitors tsa RNase li lokela ho ekeletsoa nakong ea pele-strand synthesis reaction ka pel'a buffer le agent e fokotsang (joaloka DTT), hobane ts'ebetso pele ho cDNA synthesis e senya inhibitor, kahoo e lokolla RNase e tlamiloeng e ka senyang RNA.Li-protein RNase inhibitors li thibela feela ho senyeha ha RNA ka RNase A, B, C, 'me ha li thibele RNase letlalong, kahoo e-ba hlokolosi hore u se ke ua hlahisa RNase ho tloha menoaneng ea hau ho sa tsotellehe tšebeliso ea li-inhibitors tsena.

Reverse transcriptase e susumetsa phetoho ea RNA ho cDNA.Ka bobeli M-MLV le AMV li na le ts'ebetso ea khale ea RNaseH ho kenyelletsa tšebetso ea tsona ea polymerase.Ketsahalo ea RNaseH le mesebetsi ea polymerase li qothisana lehlokoa le tse ling bakeng sa strand e nyalisitsoeng e entsoeng pakeng tsa thempleite ea RNA le DNA primer kapa cDNA extension strand, le ho theola RNA strand ho RNA:DNA complex.Thempleite ea RNA e senyehileng ke ts'ebetso ea RNaseH e ke ke ea hlola e sebetsa e le substrate e sebetsang bakeng sa motsoako oa cDNA, e fokotsang chai le bolelele ba motsoako oa cDNA.Ka hona, ho ka ba molemo ho felisa kapa ho fokotsa haholo ts'ebetso ea RNaseH ea reverse transcriptase..

SuperScript Ⅱ reverse transcriptase, RNaseH- MMLV reverse transcriptase le thermoScript reverse transcriptase, RNaseH- AMV, e ka fumana chelete e ngata le bolelele bo felletseng ba cDNA ho feta MMLV le AMV.Kutloelo-bohloko ea RT-PCR e tla angoa ke bongata ba motsoako oa cDNA.ThermoScript e bonolo haholo ho feta AMV.Boholo ba lihlahisoa tsa RT-PCR bo lekantsoe ke bokhoni ba reverse transcriptase ho kopanya cDNA, haholo ha ho kopanngoa li-cDNA tse kholoanyane.Ha ho bapisoa le MMLV, SuperScripⅡ e ekelitse haholo lihlahisoa tse telele tsa RT-PCR.RNaseH-reverse transcriptase le eona e ekelitse thermostability, kahoo karabelo e ka etsoa ka mocheso o phahameng ho feta o tloaelehileng oa 37-42°C.Tlas'a maemo a tlhahiso ea tlhahiso, sebelisa oligo(dT) primer le 10 μCi ea [α-P]dCTP.Kakaretso ea lihlahisoa tsa strand ea pele e ne e baloa ho sebelisoa mokhoa oa TCA precipitation.cDNA ea bolelele bo felletseng e ile ea hlahlobjoa ho sebelisoa lihlopha tse hlophiloeng ka boholo tse chekiloeng le ho baloa ka gel ea alkaline ea agarose.

3. Phahamisa mocheso oa incubation bakeng sa mongolo o furallang:

Mocheso o phahameng oa incubation o thusa ho bula sebopeho sa bobeli sa RNA, ho eketsa chai ea karabelo.Bakeng sa litempele tse ngata tsa RNA, ho kenya RNA le li-primers ho 65 ° C ntle le buffer kapa letsoai, ho lateloa ke ho pholisa ka potlako holim'a leqhoa ho tla felisa mekhoa e mengata ea bobeli le ho lumella li-primers ho tlama.Leha ho le joalo, litempele tse ling li ntse li e-na le meaho ea bobeli, le kamora ho felloa ke mocheso.Katoloso ea litempele tsena tse thata e ka etsoa ho sebelisoa ThermoScript Reverse Transcriptase le ho beha sengoloa se ka morao mochesong o phahameng ho ntlafatsa matlafatso.Lithempereichara tse phahameng tsa incubation li ka boela tsa eketsa ho khetheha, haholo ha li-primers tsa gene-specific (GSP) li sebelisoa bakeng sa motsoako oa cDNA (sheba Khaolo ea 3).Haeba u sebelisa GSP, etsa bonnete ba hore Tm ea li-primers e tšoana le mocheso o lebeletsoeng oa ho incubation.Se ke oa sebelisa oligo(dT) le li-primers tse sa reroang ho feta 60°C.Li-primers tse sa fetoheng li hloka ho futhumatsoa ho 25°C metsotso e 10 pele li nyolohela ho 60°C.Ntle le ho sebelisa mocheso o phahameng oa reverse transcript, ho khetheha ho ka ntlafatsoa ka ho fetisa motsoako oa RNA/primer ka kotloloho ho tloha mochesong oa 65 ° C ho ea ho mocheso o ka morao oa transcription incubation le ho eketsa motsoako o futhumetseng oa 2× (cDNA hot-start synthesis) .Mokhoa ona o thusa ho thibela "intermolecular base pairing" e hlahang mochesong o tlase.Phetoho e ngata ea mocheso e hlokahalang bakeng sa RT-PCR e ka nolofatsoa ka ho sebelisa sebaesekele se futhumatsang.

Tth thermostable polymerase e sebetsa joalo ka DNA polymerase boteng ba Mg2+ le joalo ka RNA polymerase ka pel'a Mn2+.E ka bolokoa e futhumetse ka mocheso o phahameng oa 65°C.Leha ho le joalo, ho ba teng ha Mn2 + nakong ea PCR ho fokotsa botšepehi, e leng se etsang hore Tth polymerase e se ke ea tšoaneleha bakeng sa amplification e phahameng ka ho fetisisa, e kang cloning ea cDNA.Ntle le moo, Tth e na le ts'ebetso e tlase e tlase ea mongolo, e fokotsang kutlo, 'me, kaha reverse transcript le PCR li ka etsoa ka enzyme e le' ngoe, ho laola maikutlo ntle le ho ngoloa ka morao ho ke ke ha sebelisoa ho bapisa lihlahisoa tsa cDNA tsa amplification le DNA e silafatsang ea genomic.Lihlahisoa tsa amplification li ile tsa aroloa.

4. Li-additives tse khothalletsang ho ngoloa ka morao:

Li-additives tse kenyelletsang glycerol le DMSO li kenyelletsoa karabelong ea pele-strand synthesis, e ka fokotsang botsitso ba nucleic acid double-strand le ho lokolla sebopeho sa bobeli sa RNA.Ho fihla ho 20% glycerol kapa 10% DMSO e ka eketsoa ntle le ho ama tšebetso ea SuperScript II kapa MMLV.AMV e ka boela ea mamella ho fihlela ho 20% ea glycerol ntle le tahlehelo ea mosebetsi.Bakeng sa ho holisa kutlo ea RT-PCR ho SuperScriptⅡ reverse transcript reaction, 10% glycerol e ka eketsoa le ho eketsoa ho 45°C.Haeba 1/10 ea sehlahisoa sa reverse transcription reaction e eketsoa ho PCR, joale mahloriso a glycerol karabelong ea amplification ke 0.4%, e sa lekaneng ho thibela PCR.

5. Phekolo ea RNaseH:

Kalafo ea karabelo ea motsoako oa cDNA ka RNaseH pele ho PCR e ka eketsa kutlo.Bakeng sa litempele tse ling, ho nahanoa hore RNA ho cDNA synthesis reaction e thibela ho tlama ha lihlahisoa tsa amplification, moo kalafo ea RNaseH e ka eketsang kutlo.Ka kakaretso, kalafo ea RNaseH ea hlokahala ha ho holisa lithempleite tse shebiloeng tsa cDNA tsa bolelele bo felletseng, joalo ka kopi e tlase ea tuberous scheresis II.Bakeng sa template ena e thata, kalafo ea RNaseH e ntlafalitse lets'oao le hlahisitsoeng ke SuperScript II kapa cDNA e entsoeng ka AMV.Bakeng sa maikutlo a mangata a RT-PCR, phekolo ea RNaseH ke ea boikhethelo, hobane mohato oa PCR denaturation ho 95 ° C ka kakaretso o hlahisa RNA ho RNA: DNA complex.

6. Ntlafatso ea Mokhoa o Monyane oa ho Lemoha RNA:

RT-PCR e thata haholo ha ho na le RNA e nyane feela.Glycogen e kenyellelitsoeng e le sejari nakong ea ho itšehla thajana ea RNA e thusa ho eketsa chai ea lisampole tse nyane.RNase-free glycogen e ka eketsoa ka nako e le 'ngoe le ho eketsa Trizol.Glycogen e qhibiliha ka metsing 'me e ka bolokoa mokhahlelong oa metsi ka RNA ho thusa ho na ka mor'a moo.Bakeng sa lisampole tse ka tlase ho 50 mg ea lisele kapa lisele tse 106 tse hlahisitsoeng, mohopolo o khothaletsoang oa RNase-free glycogen ke 250 μg/ml.

Ho kenyelletsa acetylated BSA ho karabelo e ka morao ea transcript ho sebelisa SuperScript II ho ka eketsa kutlo, mme bakeng sa RNA e nyane, ho fokotsa palo ea SuperScript II le ho eketsa li-unit tse 40 tsa RNaseOut nuclease inhibitor ho ka eketsa boemo ba ho lemoha.Haeba glycogen e sebelisoa ts'ebetsong ea ho itšehla thajana ea RNA, e ntse e khothaletsoa ho eketsa BSA kapa RNase inhibitor ha u sebelisa SuperScript II bakeng sa karabelo e ka morao ea transcript.

Ka mohlala, Eketsa RT-PCR e ikhethang

1. Asynthesis ea CND:

Pele-strand cDNA synthesis e ka qalisoa ho sebelisoa mekhoa e meraro e fapaneng, e tobileng ea eona e amang palo le mofuta oa cDNA e entsoeng.

Mokhoa o sa reroang oa ho qala e ne e le o fokolang haholo ho mekhoa e meraro.Li-Primer anneal libakeng tse ngata ho pholletsa le sengoloa, li hlahisa li-cDNA tse khutšoane, tse bolelele bo sa fellang.Mokhoa ona o sebelisoa khafetsa ho fumana tatellano ea ho qetela ea 5 le ho fumana cDNA ho litempele tsa RNA tse nang le libaka tsa sebopeho sa bobeli kapa ka libaka tsa ho felisa tse ke keng tsa phetoa ke reverse transcriptase.Ho fumana cDNA e telele ka ho fetisisa, karo-karolelano ea li-primers ho RNA sampoleng e 'ngoe le e 'ngoe ea RNA e hloka ho khethoa ka matla.Khokahano ea ho qala ea li-primers e sa fetoheng e ne e tloha ho 50 ho isa ho 250 ng ka karabelo ea 20 μl.Kaha cDNA e entsoe ho tsoa ho kakaretso ea RNA e sebelisang li-primers tse sa reroang ke ribosomal RNA, poly(A)+RNA hangata e khethoa joalo ka thempleite.

Li-primer tsa Oligo(dT) li tobile ho feta li-primers tse sa fetoheng.E kopana le mohatla oa poly(A) o fumanoang qetellong ea 3′ ea boholo ba li-mRNA tsa eukaryotic.Hobane poly(A)+ RNA e batla e le 1% ho isa ho 2% ea kakaretso ea RNA, palo le ho rarahana ha cDNA li tlase haholo ho feta li-primers tse sa fetoheng.Ka lebaka la boikhethelo ba eona bo phahameng, oligo(dT) ka kakaretso ha e hloke ho ntlafatsoa ha karo-karolelano ea RNA ho li-primers le khetho ea poly(A)+.Ho kgothaletswa ho sebedisa 0.5μg oligo(dT) ho ya ka 20μl reaction system.oligo(dT)12-18 e loketse boholo ba RT-PCR.ThermoScript RT-PCR System e fana ka oligo(dT)20 ka lebaka la botsitso ba eona bo betere ba mocheso bakeng sa mocheso o phahameng oa incubation.

Gene specific primers (GSP) ke li-primers tse ikhethileng ka ho fetesisa bakeng sa mohato oa khatiso o ka morao.GSP ke oligonucleotide ea antisense e ka ikamahanyang ka ho toba le tatellano ea sepheo sa RNA, ho fapana le li-primers kapa oligo(dT), tse amang li-RNA tsohle.Melao e ts'oanang e sebelisitsoeng ho rala li-primer tsa PCR e sebetsa ho moralo oa GSP ho karabelo ea morao-rao ea mongolo.GSP e ka ba tatellano e ts'oanang le ea sehokelo sa amplification se fihlang pheletsong ea 3'-boholo ba mRNA, kapa GSP e ka etsoa ho theola noka ea sehlomathiso se ka morao.Bakeng sa lithuto tse ling tse matlafalitsoeng, ho na le li-primer tsa antisense tse fetang bonngoe tse lokelang ho etsoa bakeng sa RT-PCR e atlehileng hobane sebopeho sa bobeli sa sepheo sa RNA se ka thibela ho tlama li-primer.Ho khothaletsoa ho sebelisa 1 pmol antisense GSP ho 20 μl ea pele-strand synthesis reaction.

2. Phahamisa mocheso oa incubation bakeng sa mongolo o furallang:

E le hore ho sebelisoe molemo o feletseng oa ho khetheha ha GSP, reverse transcriptase e nang le thermostability e phahameng e lokela ho sebelisoa.Thermostable reverse transcriptases e ka eketsoa ka mocheso o phahameng ho eketsa matla a ho arabela.Ka mohlala, haeba GSP e hanela ho 55°C, ho khetheha ha GSP ho ke ke ha sebelisoa ka botlalo haeba AMV kapa M-MLV e sebelisoa bakeng sa khatiso e khutlisetsang morao ka stringency e tlase ea 37°C.Leha ho le joalo, SuperScript II le ThermoScript li ka arabeloa ho 50 ° C kapa ho feta, e leng se tla felisa lihlahisoa tseo e seng tse khethehileng tse hlahisoang ka mocheso o tlase.Bakeng sa lintlha tse hlakileng, motsoako oa RNA/primer o ka fetisetsoa ka kotloloho ho tloha mochesong oa 65 ° C ho ea ho mocheso o ka morao oa transcript mme oa eketsoa motsoakong o futhumetseng oa 2× reaction (cDNA synthesis hot start).Sena se thusa ho thibela li-intermolecular base pairing ka mocheso o tlase.Liphetoho tse ngata tsa mocheso tse hlokahalang bakeng sa RT-PCR li ka nolofatsoa ka ho sebelisa sebaesekele sa mocheso.

3. E fokotsa tšilafalo ea DNA:

Bothata bo ka bang teng ka RT-PCR ke tšilafalo ea DNA ea genomic ho RNA.Ho sebelisa mokhoa o motle oa ho itšehla thajana oa RNA, joalo ka Trizol Reagent, ho tla fokotsa bongata ba DNA ea genomic e silafatsang tokisetso ea RNA.Ho qoba lihlahisoa tse nkiloeng ho genomic DNA, RNA e ka phekoloa ka amplification-grade DNase I ho tlosa DNA e silafatsang pele e fetoleloa.Tšilo ea DNase I e ile ea felisoa ka ho kenya lisampole ho 2.0 mM EDTA metsotso e 10 ka 65°C.EDTA e ka chelate magnesium ion, ho thibela magnesium ion-e itšetlehileng ka RNA hydrolysis ka mocheso o phahameng.

Bakeng sa ho arola cDNA ea amplified ho tsoa ho lihlahisoa tsa amplification tsa genomic DNA, li-primers li ka etsoa hore anneal ka 'ngoe e arole li-exons.Lihlahisoa tsa PCR tse nkiloeng ho cDNA li tla ba khutšoanyane ho feta tse nkiloeng ho DNA e silafetseng ea genomic.Ho feta moo, teko ea taolo ntle le ho ngoloa ka morao e ile ea etsoa templateng ka 'ngoe ea RNA ho fumana hore na sekhechana se fanoeng se nkiloe ho genomic DNA kapa cDNA.Sehlahisoa sa PCR se fumanoeng ntle le ho ngoloa ka morao se tsoa ho genome.