Wonke umntu uthetha ngomgaqo wovavanyo lwe-qRT-PCR, uyilo lwe-primer, ukutolika kweziphumo, njl., kodwa ndicinga ukuba ndingabelana nawe ngomsebenzi wokulinga we-qRT-PCR.Incinci, kodwa imalunga neziphumo.
Phambi kokuba senze i-qRT-PCR, kufuneka sibe nokuqonda okucacileyo kwe-RNA yethu kunye neendlela zokusebenza.Ngapha koko, iinzame zethu zijolise ekufumaneni iziphumo, kunokuba sizilolonge nje.Ke phambi kokwenza i-qRT-PCR, kufuneka simisele le miba ilandelayo (eminye yayo isebenza kuphela kwi-SYBR).
1 Ngaba uqinisekile ukuba i-RNA yakho ayithotywanga?
I-NanoDrop 2000 inokubona kuphela ugxininiso kunye nokucoceka kwe-RNA, kodwa ayikwazi ukubona ukunyaniseka kwe-RNA.
Ixabiso le-RNA (i-RNA Intesity Number) lingabonakalisa ingqibelelo ye-RNA, efunyenwe yinkqubo ye-Agilent 2100 Bioanalyzer.
Umzobo we-Schematic diagram ye-RIN yamaxabiso kwiisampuli ezahlukeneyo ze-RNA (eukaryotes)
Nangona kunjalo, iilabhoratri ngokubanzi azinayo i-Agilent 2100 Bioanalyzer.Kule meko, sinokubona ngejeli ye-formaldehyde, kodwa imfuno yesixa esipheleleyo se-RNA iphezulu, ngoko ke indlela ekhawulezayo kukusebenzisa i-gel electrophoresis eqhelekileyo.Kuyimfuneko ukuba ibe kwindawo engenawo i-nuclease, ngoko ke kuyimfuneko ukuhlamba itanki ye-electrophoresis, ibhotile ye-sol, i-bracket ye-gel kunye ne-comb kunye ne-DEPC yamanzi.I-agarose nayo ayinayo i-nuclease (ukuba nje isandula ukuvulwa), kwaye i-Loading Buffer kufuneka ivulwe ngokutsha kangangoko, nge-1.2% yejeli.
Qaphela ukuba i-gel kufuneka ichithwe ngokupheleleyo, ngaphandle koko iya kubangela amabhendi angabonakaliyo, njengoko kuboniswe kwisampuli 9 kumzobo.Ukuba i-voltage iphezulu kakhulu okanye isebenza ixesha elide iya kuvelisa ubushushu kwaye ibangele ukuthotywa kwe-RNA, ngoko ke umbane kunye nexesha kufuneka zilawulwe ngokufanelekileyo.Ukongeza, ijeli ebalekayo inokuphinda igqibe ukuba ngaba kukho intsalela ye-DNA kwisampulu, kwaye ujonge ukuba kukho inani elikhulu leebhendi ezigciniweyo kwindawo yokuhambisa.
Umzobo.Ukufunyanwa kwe-Gel electrophoresis ye-RNA
2 Ngaba uqinisekile malunga nokuxinana kwe-cDNA yakho?
Amava abazalwana abakhulu kwi-laboratory kukuba i-cDNA ye-20 ul inkqubo efunyenwe yi-inversion nganye ihlanjululwe ngokuthe ngqo kwi-20X, ngelixa oodade be-post-doctoral bahlanjululwa nge-10X.Ndidla ngokuxhomekeka kwimeko.Ngenxa yokuba umgangatho we-RNA ekhankanywa ngumntu ngamnye wahlukile, umgangatho wokubuyisela umva nawo wahlukile, yaye ubugcisa bokubuyisela umva busenokungazinzi.
Ke ngalo lonke ixesha ndifumana i-cDNA ebuyileyo, ndiya kuqala ndiyihlambulule malunga namaxesha ama-3, kwaye emva koko ndisebenzise ijini yokugcina indlu ukwenza i-RT-PCR, inani lemijikelo liyimijikelo engama-25 ngokubanzi, ukuchonga uxinaniso oluthile, kwaye emva koko ndimisele i-dilution factor yokugqibela.
3 Ngaba uqinisekile ukuba ii-primers zakho zilula ukuzisebenzisa?
Inokudlula kwigophe lokunyibilika kwe-qRT-PCR, kodwa oku kusabiza imali.Kwiilabhoratri ezingenayo imali eninzi, xa befumana iiprimers ezininzi, banokusebenzisa i-RT-PCR eqhelekileyo ukuze babone ukuba ngaba ibhendi enye kunye nokuchonga ukucaciswa kwee-primers.Ukuba i-laboratory ayikho imali emfutshane, ukuchaneka kwazo zonke ii-primers kunokuchongwa kanye nge-melting curve.
4 Ngaba uqinisekile ukuba iimeko zakho zovavanyo zifanelekile?
I-SYBR kufuneka ikhuselwe ekukhanyeni okunamandla, ngoko ke zama ukucima isibane esingaphezulu xa ufaka i-SYBR reagent, kwaye kufuneka usebenzise ukukhanya okumnyama ukuyigqiba.
Gcina i-SYBR ku-4°C.Xa usetyenziswa, jikela phezulu nasezantsi ngobunono ukuze udibanise kakuhle ukunqanda ugwebu, kwaye ungavuthuluki ngamandla.
Abanye oodade abancinci bathanda ukudweba amanqaku kwibhodi ye-PCR ngenxa yokwesaba ukuxuba iisampuli, okungalunganga.Ngenxa yokuba iziphawuli zakho zinokuchaphazela ingqokelela yeempawu zefluorescent, ndicebisa ngokubanzi abancinci ukuba basebenzise iincwadana zovavanyo ukunceda kwinkumbulo, njengoko kubonisiwe ngezantsi.
Umzobo.Umzobo wokulayisha isampulu ye-qRT-PCR
5 Uqinisekile ukuba uyenza kakuhle?
Qiniseka ukuba unxibe iiglavu, unxibe iiglavu, unxibe iigloves, kwaye uthethe izinto ezibalulekileyo izihlandlo ezithathu.
Ukuze kuncitshiswe ukuvezwa kwe-SYBR ekukhanyeni, mna ngokobuqu ndiyathanda ukongeza itemplate kuqala, njengoko kubonisiwe kulo mfanekiso ungezantsi.Ngokwamava, ukongezwa kwesixa esincinci setemplate kunokwenzeka ukuba kubangele iimpazamo zesampulu.Ngoko ke, ukuze kuncitshiswe impazamo ebangelwa ukongeza inani elincinci le template, ndidla ngokuphinda kabini isampuli kwakhona, kwaye kabini inani xa ukongeza isampuli ukunciphisa inani le-H2O2 elongezelelweyo.
Umzobo.Idayagram ecwangcisiweyo yokulayisha i-qRT-PCR
Emva koko qwalasela inkqubo ye-qRT-PCR ngolu hlobo lulandelayo.
Umzobo.Idayagram yokulungiselela inkqubo ye-qRT-PCR
QAPHELA: Inkqubo yoqwalaselo kufuneka yenziwe kumkhenkce.
Emva kokongeza isampuli, ncamathelisa ifilimu yokutywina ecacileyo.Zama ukuba ungachukumisi umphezulu wefilimu yokutywina ecacileyo ngezandla zakho, vele usebenze ukusuka kwindawo kumacala omabini efilimu.Ngenxa yokuba ushicilelo lweminwe lunokuchaphazela ingqokelela yemiqondiso yefluorescent.Emva koko sebenzisa i-centrifuge ngokukhawuleza i-centrifuge ye-10 s ngesantya esiphantsi ukukhusela isampuli ukuba ingaxhonywa eludongeni.
Iimveliso ezinxulumeneyo:
Ixesha lokuposa: Apr-28-2023
