1. Ulwazi olusisiseko (ukuba ufuna ukubona inxalenye yovavanyo, nceda uthumele ngokuthe ngqo kwinxalenye yesibini)
Njengokusabela kokuphuma kwe-PCR eqhelekileyo, Ixesha lokwenyani i-PCR ijonga ikakhulu utshintsho lwesixa semveliso yokukhulisa imveliso kumjikelo ngamnye wempendulo yokukhulisa i-PCR ngexesha lokwenyani ngotshintsho lwesiginali ye-fluorescence, kwaye ngokobungakanani ihlalutya itemplate yokuqalisa ngobudlelwane phakathi kwexabiso le-ct kunye negophe eliqhelekileyo.
Iinkcukacha ezithile ze-RT-PCR ziisiseko, i-fluorescence thresholdkwayeCt ixabiso.
| isiseko: | Ixabiso le-fluorescence yomjikelezo we-3-15 yisiseko (isiseko), esibangelwa yimpazamo yezihlandlo ezithile zokulinganisa. |
| Umda (umda): | Ibhekisa kumda wokubona i-fluorescence ebekwe kwindawo efanelekileyo kwindawo yokukhula okubonakalayo kwegophe lokukhulisa, ngokuqhelekileyo amaxesha ali-10 ukutenxa okusemgangathweni kwesiseko. |
| Ixabiso le-CT: | Linani lemijikelo ye-PCR xa ixabiso le-fluorescence kwityhubhu nganye yokusabela ifikelela embundwini. Ixabiso le-Ct lihambelana ngokuhlukileyo kwisixa sethemplethi yokuqala. |
Iindlela eziqhelekileyo zokuleyibhela ze-RT-PCR:
| indlela | inzuzo | ukusilela | umda wesicelo |
| SYBR GreenⅠ | Ukusebenziseka okubanzi, ubuthathaka, ixabiso elincinci kwaye lilungele | Iimfuno ze-Primer ziphezulu, zixhomekeke kwiibhendi ezingezizo ezodwa | Ilungele uhlalutyo lobungakanani beentlobo ezahlukeneyo zofuzo ekujoliswe kuzo, uphando malunga nembonakalo yemfuza, kunye nophando malunga nezilwanyana kunye nezityalo eziphinda ziphinde ziphinde zenziwe. |
| TaqMan | Ukuchaneka okulungileyo kunye nokuphindaphinda okuphezulu | Ixabiso liphezulu kwaye lifanelekile kuphela kwiinjongo ezithile. | Ukufunyaniswa kwe-pathogen, uphando lwemfuza yokumelana neziyobisi, ukuhlolwa kokusebenza kwechiza, ukuxilongwa kwezifo zemfuzo. |
| ibhakana yemolekyuli | Ukuchaneka okuphezulu, i-fluorescence, imvelaphi ephantsi | Ixabiso liphezulu, lifanelekile kuphela kwinjongo ethile, ukuyila kunzima, kwaye ixabiso liphezulu. | Uhlalutyo oluthile lwe-gene, uhlalutyo lwe-SNP |
2. Amanyathelo okulinga
2.1 Malunga neqela lovavanyo- kufuneka kubekho amaqula amaninzi kwiqela, kwaye kufuneka kubekho ukuphindaphinda kwezinto eziphilayo.
| ① | Ulawulo olungenanto | Isetyenziselwa ukubona imeko yokukhula kweeseli kwimifuniselo |
| ② | Ulawulo olubi i-siRNA (ulandelelwano lwe-siRNA olungangqalanga) | Bonisa ubunjani besenzo se-RNAi.I-siRNA inokubangela impendulo yoxinzelelo olungelulo ngokuthe ngqo kwi-concentration ye-200nM. |
| ③ | Ulawulo lwe-Reagent yoTshintsho | Kungabandakanyi ubutyhefu be-reagent yokudluliselwa kwiiseli okanye umphumo wokubonakaliswa kwejeni ekujoliswe kuyo |
| ④ | i-siRNA ngokuchasene nofuzo ekujoliswe kulo | Betha imbonakalo yejini ekujoliswe kuyo |
| ⑤ (uyazikhethela) | i-siRNA entle | Isetyenziselwa ukulungisa iingxaki zesistim yovavanyo kunye neengxaki zokusebenza |
| ⑥ (uyazikhethela) | Ulawulo lweFluorescent siRNA | Ukusebenza kakuhle kokudluliselwa kweeseli kunokubonwa nge-microscope |
2.2 Imigaqo yoyilo lweprimer
| Ubungakanani beqhekeza elinyusiweyo | Ngokukhethekileyo kwi-100-150bp |
| Ubude bePrimer | 18-25bp |
| Umxholo weGC | 30%-70%, kukhethwa ukuba 45%-55% |
| Tm ixabiso | 58-60℃ |
| Ulandelelwano | Gwema i-T / C eqhubekayo;I-A/G iyaqhubekeka |
| 3 ekupheleni ulandelelwano | Kuphephe i-GC etyebileyo okanye i-AT etyebileyo;isiseko setheminali kukhethwa ukuba yi-G okanye C;kungcono ukuphepha uT |
| Ukuhambelana | Gwema ulandelelwano oluhambelanayo lweziseko ezingaphezulu kwe-3 ngaphakathi kwe-primer okanye phakathi kwee-primers ezimbini |
| Ukuchaza ngokuthe ngqo | Sebenzisa uphendlo oluqhumayo ukuze uqinisekise ubume be-primer |
①I-SiRNA iluhlobo oluthile, kwaye ukulandelelana kweentlobo ezahlukeneyo kuya kwahluka.
I-②SiRNA ifakwe kwi-powder eyomileyo, enokuthi igcinwe ngokuzinzileyo kwiiveki ezi-2-4 kwiqondo lokushisa.
2.3 Izixhobo okanye ii-reagents ekufuneka zilungiswe kwangethuba
| Iprimer (isalathiso sangaphakathi) | Ukuquka ukuya phambili kunye nokubuyisela umva ezimbini |
| Iiprimers (igene ekujoliswe kuyo) | Ukuquka ukuya phambili kunye nokubuyisela umva ezimbini |
| Ujoliso Si RNA (imicu emi-3) | Ngokubanzi, inkampani iya kwenza imicu emi-3, kwaye emva koko ukhethe enye kwezintathu nge-RT-PCR |
| UTshintsho Kit | Lipo2000 njl. |
| RNA Rapid Extraction Kit | Ukukhutshwa kwe-RNA emva kokudluliselwa |
| Rapid Reverse Transcription Kit | ye-cDNA synthesis |
| Ikhithi yoKwandiswa kwePCR | I-2 × Super SYBR eluhlaza qPCR Master Mix |
2.4 Ngokumalunga nemiba ekufuneka ithathelwe ingqalelo kumanyathelo ovavanyo athile:
① inkqubo yokudluliselwa kwe-siRNA
1. Ukucoca, unokukhetha i-plate ye-24-well plate, i-12-well plate okanye i-6-well plate (umyinge we-RNA woxinaniso olucetywayo kwiqula ngalinye le-plate ye-24-well malunga ne-100-300 ng / uL), kwaye ubuninzi bokugqithiswa kweeseli bufikelela kwi-60 % -80% okanye njalo.
2. Amanyathelo okutshintshela kunye neemfuno ezithile zihambelana ngokungqongqo nemiyalelo.
3. Emva kokudluliselwa, iisampuli zingaqokelelwa kwiiyure ze-24-72 zokufumanisa i-mRNA (RT-PCR) okanye ukufumanisa iprotheni kwiiyure ze-48-96 (WB)
② Inkqubo yokutsalwa kwe-RNA
1. Thintela ukosuleleka zii-exogenous enzymes.Ikakhulu ibandakanya ukunxiba iimaski kunye neiglavu ngokungqongqo;usebenzisa iingcebiso zepipette ezintsholongwane kunye neetyhubhu ze-EP;amanzi asetyenzisiweyo kuvavanyo kufuneka abe RNase-Free.
2. Kucetyiswa ukuba wenze kabini njengoko kucetyisiwe kwikhithi yokukhupha ngokukhawuleza, eya kuphucula ngokwenene ukucoceka kunye nesivuno.
3. Ulwelo lwenkunkuma mayingachukumisi ikholamu yeRNA.
③ Ubalo lwe-RNA
Emva kokuba i-RNA ikhutshwe, inokulinganiswa ngokuthe ngqo ngeNanodrop, kwaye ubuncinane bokufunda bunokuba buphantsi njenge-10ng / ul.
④Umva inkqubo yokukhutshelwa
1. Ngenxa yokuqonda okuphezulu kwe-RT-qPCR, ubuncinane i-3 parallel wells kufuneka yenziwe kwisampuli nganye ukukhusela i-Ct elandelayo ukuba ihluke kakhulu okanye i-SD ibe inkulu kakhulu kuhlalutyo lwamanani.
2. Musa ukuba ngumkhenkce kwaye unyibilikise i-Master mix ngokuphindaphindiweyo.
3. Umbhobho/umngxuma ngamnye mawutshintshwe ngencam entsha!Musa ukusebenzisa ngokuqhubekayo incam yepipette efanayo ukongeza iisampuli!
4. Ifilimu efakwe kwi-plate ye-96-well-plate emva kokufaka isampuli kufuneka ihlanjululwe ngeplate.Kungcono ukuyifaka kwi-centrifuge phambi kokuyibeka kumatshini, ukuze ulwelo oluseludongeni lombhobho lukwazi ukuhla luze lususe amaqamza omoya.
⑤Uhlalutyo oluqhelekileyo lwegophe
| Akukho xesha lokukhula kwe-logarithmic | Uxinzelelo olunokwenzeka kakhulu lwetemplate |
| Akukho xabiso leCT | Amanyathelo angalunganga okufumana iimpawu ze-fluorescent; ukuthotywa kwe-primers okanye i-probes - ingqibelelo yayo inokubonwa nge-PAGE electrophoresis; isixa esinganelanga setemplate; ukuthotywa kweetemplates - ukuphepha ukungeniswa kokungcola kunye nokukhenkcezwa okuphindaphindiweyo kunye nokunyibilika ekulungiseleleni isampuli; |
| Ct>38 | Ukusebenza kokukhulisa okuphantsi;Imveliso yePCR inde kakhulu;izinto ezahlukeneyo zokusabela zithotywa |
| Igophe lokukhulisa umgca | Iiprobe zingonakala ngokuyinxenye ngokuphinda-phinda komjikelo wokunyibilikisa okanye ukuba sesichengeni sokukhanya ixesha elide. |
| Umahluko kwimingxuma ephindwe kabini mkhulu kakhulu | Isisombululo sokusabela asinyibiliki ngokupheleleyo okanye isisombululo sokusabela asixutywanga;Ibhafu ye-thermal yesixhobo se-PCR ingcoliswe zizinto ze-fluorescent |
2.5 Malunga nohlalutyo lwedatha
Uhlalutyo lwedatha lwe-qPCR lunokwahlulwa lube ngumlinganiselo onxulumeneyo kunye nobungakanani obupheleleyo.Umzekelo, iiseli kwiqela lonyango xa kuthelekiswa neeseli kwiqela lolawulo,
Mangaphi amaxesha i-mRNA ye-X itshintsha, oku kumlinganiselo ohambelanayo;kwinani elithile leeseli, i-mRNA yejini X
Zingaphi iikopi ezikhoyo, olu luqikelelo olupheleleyo.Ngokuqhelekileyo eyona nto siyisebenzisa kakhulu kwilabhoratri yindlela yobungakanani obunxulumeneyo.Ngesiqhelo,indlela ye-2-ΔΔctisetyenziswa kakhulu kwimifuniselo , ngoko ke kuphela le ndlela iyakwaziswa ngokweenkcukacha apha.
I-2-ΔΔct indlela: Isiphumo esifunyenweyo ngumahluko ekubonakalisweni kwejini ekujoliswe kuyo kwiqela lokulinga ngokumalunga nejene ekujoliswe kulo kwiqela lolawulo.Kuyafuneka ukuba ulwandiso oluchanekileyo lwejeni ekujoliswe kulo kunye nejene yereferensi yangaphakathi isondele kwi-100%, kwaye ukutenxa okunxulumeneyo makungadluli kwi-5%.
Indlela yokubala imi ngolu hlobo lulandelayo:
Δct iqela lolawulo = ixabiso lejene ekujoliswe kuyo kwiqela lolawulo - ct ixabiso lejene yereferensi yangaphakathi kwiqela lolawulo
Δct iqela lovavanyo = ixabiso lejeni ekujoliswe kuyo kwiqela lovavanyo - ct ixabiso lejene yereferensi yangaphakathi kwiqela lovavanyo
ΔΔct=Δct yovavanyo iqela-Δct iqela lolawulo
Okokugqibela, bala umahluko ophindaphindiweyo kwinqanaba lokubonisa:
Tshintsha i-Gold=2-ΔΔct (ehambelana nomsebenzi we-excel AMANDLA)
Iimveliso eziyeleleneyo:
Ixesha lokuposa: May-20-2023
