Ⅰ. 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 e lokela ho netefatsa bonyane kakaretso e telele 'me ha e na li-inhibitors tse se nang li-enzyme tsa ho rekota, tse kang EDTA kapa SDS.Boleng ba RNA bo etsa qeto ea boleng bo phahameng ba tlhahisoleseding ea tatelano eo o ka e ngollang ho cDNA.Mokhoa o akaretsang oa tlhoekiso ea RNA ke mokhoa oa mohato oa ho sebelisa isoocyanate/acidophenol.E le ho thibela tšilafalo ea RNase, RNA e arohaneng le sampole e ruileng ka RNase (joaloka manyeme) e hloka polokelo ea formaldehyde ho boloka RNA ea boleng bo holimo, e leng eona le ho feta bakeng sa polokelo ea nako e telele.RNA e ntšitsoeng sebeteng sa rat e ne e senyehile ka mor'a beke e le 'ngoe ea polokelo ka metsing, ha RNA e ntšitsoeng ho spleen ea rat e ile ea lula e tsitsitse ka mor'a lilemo tse tharo tsa polokelo ka metsing.Ho feta moo, lingoloa tse kholo ho feta 4kb li na le maikutlo a matle ho latela ho senyeha ha RNase ho feta lingoliloeng tse nyane.E le ho eketsa botsitso ba sampole ea polokelo ea RNA, RNA e ka qhibiliha ka methalmamine ea ion, 'me e bolokiloe -70 °C.Thylide e sebelisitsoeng ho boloka RNA ha ea tlameha ho ba le lintho tse fapaneng tse theolang RNA.RNA, e nkiloeng ho manyeme, e ka bolokoa ka methalmamine bonyane selemo se le seng.Ha u se u itokiselitse ho sebelisa RNA, u ka sebelisa mekhoa e latelang ho fokotsa RNA: eketsa NaCl ho 0.2m le makhetlo a 4 ho feta molumo oa ethanol, sebaka sa mocheso oa kamore metsotso e 3-5, le 10,000 × g centrifugal metsotso e 5.

2. Sebelisa reverse transcriptase ntle le ts'ebetso ea RNaseH (RNaseH-):

RNase inhibitors hangata e eketsoa ho fetola karabelo ea transcript ho eketsa bolelele le chai ea motsoako oa cDNA.RNase inhibitor e eketsoa karabelong ea ketane ea pele ea ketane ka pel'a li-buffers le li-agent tse fokotsang joalo ka DTT hobane ts'ebetso ea pre-cDNA synthesis e theola inhibitor, ka hona e lokolla li-RNase tse tlamiloeng tse theolang RNA.Protein RNase inhibitor e thibela feela ho senyeha ha RNA ka RNase A, B, C, 'me ha e thibele RNase letlalong, kahoo ho lokela ho nkoa tlhokomelo e le hore u se ke ua kenyelletsa RNases ho tloha menoana 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.Ts'ebetso ea RNaseH e qothisana lehlokoa le ts'ebetso ea polymerase bakeng sa likhoele tsa heterozygous tse entsoeng lipakeng tsa litempele tsa RNA le li-primer tsa DNA kapa likhoele tsa katoloso ea cDNA, 'me e theola RNA: likhoele tsa RNA ka har'a DNA complexes.Lithempleite tsa RNA tse sentsoeng ke tšebetso ea RNaseH li ke ke tsa hlola li sebelisoa e le li-substrates tse sebetsang hantle bakeng sa motsoako oa cDNA, ho fokotsa chai le bolelele ba motsoako oa cDNA.Kahoo ho felisa kapa ho fokotsa haholo ts'ebetso ea RNaseH ea reverse transcriptase e ka ba molemo o moholo.

SuperScriptⅡ reverse transcriptase, MMLV reverse transcriptase ea RNaseH- le thermoScript reverse transcriptase, AMV ea RNaseH- e hlahisitse cDNA e telele ho feta MMLV le AMV.Kutloelo-bohloko ea RT-PCR e angoa ke bongata ba cDNA e entsoeng.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 etsoa cloning li-Cdna tse kholoanyane.Ha ho bapisoa le MMLV, SuperScripⅡ e ekelitse haholo lihlahisoa tse telele tsa RT-PCR.RNaseH-'s reverse transcriptase e boetse e eketsa botsitso ba mocheso, kahoo karabelo e ka etsoa ka mocheso o phahameng ho feta o tloaelehileng oa 37-42 ℃.Tlas'a maemo a tlhahiso ea tlhahiso, oligo(dT) primers le 10μCi [alpha-p]dCTP li ile tsa sebelisoa.Kakaretso ea tlhahiso ea ketane ea pele e ne e baloa ho sebelisoa mokhoa oa TCA oa pula.cDNA ea bolelele bo felletseng e ile ea hlahlobjoa ho sebelisoa ho tlosoa ha likhoele tse hlophisitsoeng ka boholo le ho baloa ka gel ea alkaline ea agarose.

3. Eketsa mocheso oa ho boloka mocheso oa mongolo o ka morao:

Mocheso o phahameng oa ho tšoara o thusa ho bula sebopeho sa bobeli sa RNA le ho eketsa chai ea karabelo.Bakeng sa li-templates tse ngata tsa RNA, ho tšoara RNA le primer ho 65 ° C ntle le buffer kapa letsoai ebe o li pholisa ka potlako holim'a leqhoa ho 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 sebopeho sa bobeli, esita le ka mor'a denaturation ea mocheso.Ho holisa litempele tsena tse thata ho ka etsoa ho sebelisoa ThermoScript reverse transcriptase le ka ho beha reverse transcriptase reaction lithempereichareng tse phahameng ho ntlafatsa matlafatso.Lithempereichara tse phahameng li ka boela tsa eketsa ho khetheha, haholo-holo ha cDNA synthesis e etsoa ho sebelisoa liphatsa tsa lefutso (GSPS) (sheba Khaolo ea 3).Haeba u sebelisa GSP, etsa bonnete ba hore boleng ba Tm ba primer bo tšoana le mocheso o lebelletsoeng oa ho ts'oara.Se ke oa sebelisa oligo(dT) le li-primers tse sa reroang ho feta 60 ℃.Li-primers tse sa fetoheng li hloka ho ts'oaroa ka 25 ℃ metsotso e 10 pele li nyolohela ho 60 ℃.Ntle le ho sebelisa lithempereichara tse phahameng ka ho fetesisa, boits'oaro bo ka ntlafatsoa ka ho fetisetsa motsoako oa RNA / primer ka kotloloho ho tloha mochesong oa 65 ℃ oa denaturing ho ea ho mocheso o ts'oanang oa mongolo le ho eketsa motsoako oa karabelo oa 2 × (cDNA thermal initiation synthesis).Mokhoa ona o thusa ho thibela "intermolecular base pairing" e hlahang mochesong o tlase.Ho sebelisa sesebelisoa sa PCR ho nolofatsa li-switches tse ngata tsa mocheso tse hlokahalang bakeng sa RT-PCR.

Tth mocheso o tsitsitseng oa polymerase o sebetsa e le DNA polymerase ka pel'a Mg2 + le RNA polymerase ka pel'a Mn2 +.E khona ho boloka mocheso ho fihlela ho 65 ℃.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 cDNA cloning.Ho feta moo, Tth ha e sebetse hantle ha ho ngoloa ka morao, e leng ho fokotsang kutlo, 'me kaha enzyme e le' ngoe e ka etsa reverse transcript le PCR, ho laola liketso ntle le ho rekolla morao ho ke ke ha sebelisoa ho khetholla lihlahisoa tse matlafalitsoeng tsa cDNA ho tsa DNA e silafetseng.

4. Keketso e khothalletsang ho ngoloa ka morao:

Ho eketsoa ha li-additives, ho kenyelletsa le glycerin le DMSO, ho karabelo ea pele ea ketane ea synthesis ho ka fokotsa botsitso ba nucleic acid double strand le ho lokolla sebopeho sa bobeli sa RNA.Ho fihla ho 20% glycerin kapa 10% DMSO e ka eketsoa ntle le ho ama tšebetso ea SuperScriptⅡ kapa MMLV.AMV e ka boela ea mamella ho fihla ho 20% ea glycerol ntle le ho fokotsa mosebetsi.Ho eketsa kutloelo-bohloko ea RT-PCR ho SuperScriptⅡ reverse transcript reaction, 10% glycerol e ka eketsoa le ho kenngoa ka 45 ℃.Haeba 1/10 ea sehlahisoa sa retrotranscription-reaction e eketsoa ho PCR, mahloriso a glycerol karabelong ea amplification ke 0.4%, e sa lekaneng ho thibela PCR.

5. Ts'ebetso ea RNaseH:

Sensitivity e ka ntlafatsoa ka ho phekola karabelo ea motsoako oa cDNA ka RNaseH pele ho PCR.Bakeng sa litempele tse ling, ho nahanoa hore RNA ho cDNA synthesis reaction e thibela ho tlama ha lihlahisoa tse holisitsoeng, moo kalafo ea RNaseH e ka eketsang kutloelo-bohloko.Ka kakaretso, kalafo ea RNaseH ea hlokahala bakeng sa ho holisa thempleite e batlang e le telele e felletseng ea cDNA, joalo ka tuberous scheresisⅡ e nang le kopi e tlase.Bakeng sa template ena e thata, RNaseH e ntlafalitse lets'oao le hlahisitsoeng ke cDNA e entsoeng ke SuperScriptⅡ kapa AMV.Bakeng sa karabelo e ngata ea RT-PCR, kalafo ea RNaseH ke ea boikhethelo hobane mohato oa 95 ℃ o kentsoeng PCR denaturation hangata o hlahisa RNA ho tsoa ho RNA: DNA complex.

6. Mekhoa e ntlafetseng ea ho lemoha palo e nyenyane ea RNA:

RT-PCR e thata haholo ha ho na le RNA e nyane feela.Ho eketsoa ha glycogen joalo ka sejari nakong ea karohano ea RNA ho thusa ho eketsa chai ea lisampole tse nyane.Glycogen e se nang RNase e ka eketsoa ka nako e le 'ngoe le Trizol.Glycogen e qhibiliha ka metsing 'me e ka lula e le mothating oa metsi le RNA ho thusa ho na ha pula e tlang.Khokahano e khothaletsoang ea RNase-free glycogen ke 250μg/ml bakeng sa lisampole tse ka tlase ho 50mg ea lisele kapa lisele tse 106 tse hlahisitsoeng.

Keketso ea acetylated BSA ho khutlisa karabelo ea transcript e sebelisa SuperScriptⅡ e ka eketsa kutlo, mme bakeng sa RNA e nyane, ho fokotsa palo ea SuperScriptⅡ le ho eketsa li-unit tse 40 tsa RnaseOut nuclease inhibitor ho ka ntlafatsa boemo ba ho lemoha.Haeba glycogen e sebelisoa karohanong ea RNA, ho eketsoa ha BSA kapa RNase inhibitors ho fetola maikutlo a transcript ho sebelisa SuperScriptⅡ ho ntse ho khothaletsoa.

Ⅱ. Eketsa ho khetheha ha RT-PCR

1. cNDA synthesis:

Mekhoa e meraro e fapaneng e ka sebelisoa ho qala motsoako oa cDNA oa pele, 'me mokhoa o ikhethileng oa mokhoa o mong le o mong o ama palo le mofuta oa cDNA e entsoeng.

Mokhoa o sa reroang oa ho qala ke o fokolang ho feta mekhoa e meraro.Li-primers li kenngoa libakeng tse ngata ho pholletsa le sengoloa ho hlahisa cDNA e khutšoanyane, e sa fellang.Hangata mokhoa ona o sebelisoa ho fumana li-terminal tsa 5′ le cDNA ho tsoa ho litempele tsa RNA tse nang le libaka tsa meralo ea bobeli kapa ka libaka tsa ho felisa tse reverse transcriptase li ke keng tsa ikatisa.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 pele ea li-primers tse sa reroang e tloha ho 50 ho isa ho 250ng ho sistimi ea karabelo ea 20μl.Hobane cDNA e entsoeng ho tsoa ho kakaretso ea RNA e sebelisang li-primers tse sa reroang haholo-holo ke ribosomal RNA, poly(A)+RNA hangata e khethoa joalo ka thempleite.

Ho qala ha Oligo(dT) ho hlakile ho feta li-primers tse sa reroang.E kopana le mohatla oa poly(A) o fumanoang qetellong ea 3′ ea mRNA liseleng tse ngata 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 ha ho ne ho sebelisoa li-primers tse sa reroang.Ka lebaka la boikhethelo ba eona bo phahameng, oligo(dT) ka kakaretso ha e hloke hore RNA e be le tekanyo ea primer 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 botle ba mocheso ebile e loketse mocheso o phahameng oa ho ts'oara.

Li-primers tsa Gene-specific (GSP) ke li-primers tse ikhethileng ka ho fetesisa bakeng sa mohato oa khatiso o khutlelang morao.GSP ke oligonucleoside ea antisense e ka kopanyang ka ho khetheha ka tatellano ea sebaka sa RNA, ho fapana le ho kopanya li-Rna kaofela joalo ka li-primers kapa oligo(dT).Melao e sebelisitsoeng ho rala li-primer tsa PCR e sebetsa hape le moahong oa karabelo ea reverse transcription reaction GSP.GSP e ka ba tatellano e ts'oanang le ea pele ea amplification e kenyellelitsoeng qetellong ea mRNA3′, kapa GSP e ka raloa hore e koalehe ho theosa le noka ka sehlahisoa sa amplification se ka morao.Bakeng sa lintho tse ling tse holisitsoeng, hoa hlokahala ho etsa moralo o fetang o le mong oa antisense bakeng sa RT-PCR e atlehileng hobane sebopeho sa bobeli sa sepheo sa RNA se ka thibela primer ho tlama.Ho khothalletsoa ho sebelisa 1pmol antisense GSP tsamaisong ea pele ea ketane ea synthesis ea 20μl.

2. Eketsa mocheso oa ho boloka mocheso oa mongolo o ka morao:

E le ho nka monyetla ka botlalo ba GSP, reverse transcriptase e nang le botsitso bo phahameng ba mocheso e lokela ho sebelisoa.Heat-stable reverse transcriptase e ka bolokoa lithempereichara tse holimo ho eketsa matla a ho arabela.Ka mohlala, haeba GSP e haneloa ka 55°C, joale ho khetheha ha GSP ha ho sebelisoe ka botlalo haeba ho ngoloa ha reverse ho etsoa ka 37°C ka matla a tlaase ho sebelisoa AMV kapa M-MLV.Leha ho le joalo, SuperScripⅡ le ThermoScript li khona ho arabela ho 50℃ kapa ho feta, e leng se felisang lihlahisoa tse sa tobang tse hlahisoang ka mocheso o tlase.Bakeng sa lintlha tse hlakileng, motsoako oa RNA / primer o ka fetisetsoa ka kotloloho ho tloha mocheso oa 65 ℃ ho ea ho mocheso o ts'oaretsoeng ka morao ka kenyelletso ea motsoako oa karabelo ea 2 x preheated (ho qala ka mocheso oa cDNA synthesis).Sena se thusa ho thibela ho kopana ha motheo pakeng tsa limolek'hule ka mocheso o tlase.Ho sebelisa sesebelisoa sa PCR ho nolofatsa liphetoho tse ngata tsa mocheso tse hlokahalang bakeng sa RT-PCR.

3. Fokotsa tšoaetso ea DNA ea genomic:

Bothata bo bong bo ka bang teng ka RT-PCR ke hore RNA e silafatsa DNA ea genomic.Tšebeliso ea mekhoa e metle ea karohano ea RNA, e kang Trizol Reagent, e fokotsa tšilafalo ea DNA ea genomic litokisetsong tsa RNA.Ho qoba lihlahisoa tse hlahisoang ho tsoa ho genomic DNA, RNA e ka alafshoa ka "amplification grade DnasⅠ" ho tlosa DNA e silafetseng pele ho phetisetsoa morao.Mehlala e ile ea bolokoa ho 65℃ ho 2.0mM EDTA bakeng sa metsotso e 10 ho felisa tšilo ea lijo ea DNaseⅠ.EDTA e chelate li-ion tsa magnesium ho thibela magnesium ion e itšetlehileng ka RNA hydrolysis e hlahang mochesong o phahameng.

Bakeng sa ho arola cDNA ea amplified ho sehlahisoa sa DNA amplification ea genome, li-primers tse anneng ka thoko le exon e arohaneng li ka etsoa.Lihlahisoa tsa PCR tse nkiloeng ho cDNA li tla ba khutšoanyane ho feta tse nkiloeng ho DNA e silafetseng ea genomic.Teko e laoloang ntle le ho ngoloa ka morao e boetse e etsoa ho thempleite ka 'ngoe ea RNA ho fumana hore na sekhechana se fanoeng se tsoa ho genomic DNA kapa cDNA.Lihlahisoa tsa PCR tse fumanoeng ha ho se na transcript li tsoa ho genome.

Sehlahisoa se Amanang

RT-PCR e bonolo^ᵀᴹKe (Mohato o le mong)

-Setsi sa mohato o le mong se nolofalletsa ho ngoloa ka morao le PCR ho etsoa ka tube e le 'ngoe.E hloka feela ho eketsa template ea RNA, li-primer tsa PCR le RNase-Free ddH₂O.

- Tlhahlobo ea nako ea sebele ea RNA e ka etsoa kapele le ka nepo.

-The Kit e sebelisa e ikhethang Foregene reverse transcription reagent le Foregene HotStar Taq DNA Polymerase e kopantsoeng le mokhoa o ikhethang oa karabelo ho ntlafatsa ka katleho katleho ea amplification le ho khetheha ha karabelo.

-Sistimi e ntlafalitsoeng ea karabelo e etsa hore karabelo e be le kutlo e phahameng ea ho lemoha, botsitso bo matla ba mocheso, le mamello e ntle.

RT Easy II(With GDNase) Master Premix For First-Strand CDNA Synthesis For Real Time PCR With GDNase

-Bokhoni bo botle ba ho tlosa gDNA, e ka tlosa gDNA ka template ka hare ho metsotso ea 2.

-Sistimi e sebetsang hantle ea ho fetolela, ho nka metsotso e 15 feela ho phethela motsoako oa cDNA ea pele.

Li-templates tse rarahaneng: litempele tse nang le litaba tse phahameng tsa GC le sebopeho sa bobeli se rarahaneng le tsona li ka khutlisoa ka katleho e phahameng.

-High-sensitivity reverse transcription system, litempele tsa pg-level le tsona li ka fumana cDNA ea boleng bo holimo.

-The reverse transcript system e na le botsitso bo phahameng ba mocheso, mocheso o nepahetseng oa ho arabela ke 42℃, 'me e ntse e na le ts'ebetso e ntle ea ho fetolela ho 50℃.