Qhov tshwj xeeb ntawm kev kuaj pom

Feem ntau, lub hom phiaj ntawm kev tsim primer yog ua kom pom tseeb ntawm PCR.Qhov no yog txiav txim siab los ntawm ntau dua lossis tsawg dua kev cuam tshuam ntawm ntau qhov sib txawv.Ib qho tseem ceeb sib txawv yog qhov sib lawv liag ntawm 3′ kawg ntawm primer.

Qhov tseem ceeb, PCR kev soj ntsuam tsim los rau qhov tshwj xeeb muaj peev xwm tswj tau qhov kev ua haujlwm siab tshaj qhov dav dav, vim tias qhov kev ntsuam xyuas tsis tsim cov khoom lag luam tsis tshwj xeeb, yog li sib tw nrog PCR reagents lossis inhibiting lub ntsiab amplification cov tshuaj tiv thaiv.

Tau kawg, qee zaum, qhov tshwj xeeb tsis yog qhov tseem ceeb tshaj plaws, piv txwv li, thaum lub hom phiaj yog txhawm rau txheeb xyuas qhov sib thooj tab sis cov kab mob sib txawv, kev tsim tshwj xeeb, kev ua kom zoo dua qub thiab cov qauv pov thawj yuav tsum muaj.

Lub melting nkhaus yog ib txoj hauv kev los ntsuam xyuas qhov tshwj xeeb ntawm amplicons, yam tsawg kawg ntawm seb puas yuav ua kom nrov rau ib lub hom phiaj.Txawm li cas los xij, nws yuav tsum tau hais qhia tias qhov kev sib tw melting tuaj yeem ua rau tsis meej pem vim tias, piv txwv li, lawv tuaj yeem cuam tshuam los ntawm kev sib xyaw ua ke ntawm cov primers tsis zoo thiab cov qauv qis.

P5 |Lub melting nkhaus qhia tau hais tias Tm hloov tau los ntawm ob qhov kev tshawb pom ntawm qhov sib txawv ntawm ob lub hom phiaj DNAs.

A. Ntawm qhov siab dua (ad)), tsis muaj qhov pom tseeb primer dimer tom qab kev ntsuas qPCR tiav.Raws li template concentration txo mus rau 50 daim ntawv luam (e), ib yam khoom tsis tshwj xeeb pib tshwm thiab dhau los ua ib yam khoom ntawm qhov qis tshaj concentration (f).

B. Qhov kev ntsuam xyuas tau sau tib Tms ntawm txhua lub hom phiaj, thiab tsis muaj qhov pom tseeb primer dimer txawm tias qhov qis tshaj (5 daim ntawv luam).Thaum siv ob txoj kev tshawb nrhiav no, tsis muaj cov khoom lag luam amplification raug kuaj pom hauv NTCs.

P5 qhia txog kev sib cais nkhaus tau nrog cov qauv uas tus qauv muaj nyob ntawm qhov sib txawv.P 5a qhia tau hais tias ntawm ob qhov qis qis tshaj plaws, Tms ntawm cov khoom lag luam amplification uas tsis yog tshwj xeeb tsim tau qis dua li ntawm cov amplicons tshwj xeeb.

Obviously, txoj kev tshawb nrhiav no tsis tuaj yeem siv tau los txheeb xyuas cov hom phiaj uas muaj nyob hauv qhov tsis tshua muaj siab.

Interestingly, NTCs, piv txwv li, cov qauv uas tsis muaj DNA txhua, tsis tau sau (tsis yog tshwj xeeb) amplification cov khoom, qhia tias keeb kwm genomic DNA tuaj yeem koom nrog hauv cov tsis tshwj xeeb amplification/polymerization.

Qee lub sij hawm xws li keeb kwm yav dhau los thiab tsis muaj qhov tshwj xeeb amplification tsis tuaj yeem kho, tab sis feem ntau nws muaj peev xwm los tsim ib txoj kev tshawb nrhiav uas tsis muaj qhov tsis muaj qhov tshwj xeeb amplification nyob rau hauv ib qho template concentration thiab NTC (P 5b).

Ntawm no, txawm kaw qhov kev nthuav dav ntawm lub hom phiaj concentration nrog Cq ntawm 35 yuav tsim ib qho kev sib cais tshwj xeeb.Ib yam li ntawd, NTCs tsis pom cov cim ntawm qhov tsis yog qhov tshwj xeeb amplification.Qee lub sij hawm, kev kuaj pom tus cwj pwm tuaj yeem nyob ntawm niam cawv, thiab tsuas yog kuaj pom tsis muaj qhov tshwj xeeb hauv cov tshuaj tsis sib xws, uas yuav cuam tshuam rau qhov sib txawv Mg2 + ntau.

Kev ruaj ntseg ntawm kev kuaj pom

Kev ua kom zoo dua ntawm Ta yog ib kauj ruam tseem ceeb hauv kev txheeb xyuas qhov tseeb thiab kev ua kom zoo ntawm qPCR nrhiav pom.Nws muab cov lus qhia ncaj qha ntawm qhov muaj zog ntawm cov primer teeb tsa los ntawm kev qhia qhov ntsuas kub (lossis qhov kub thiab txias) uas ua rau qis tshaj Cq yam tsis tau nthuav dav NTC.

Qhov sib txawv ntawm ob mus rau plaub npaug ntawm qhov rhiab heev yuav tsis yog qhov tseem ceeb rau cov neeg uas muaj mRNA siab, tab sis rau kev kuaj mob, nws yuav txhais tau tias qhov sib txawv ntawm qhov zoo thiab qhov tsis zoo.

Cov khoom Ta ntawm qPCR primers yuav txawv heev.Qee qhov kev ntsuam xyuas tsis muaj zog heev, thiab yog tias lawv tsis ua raws li qhov pom zoo Ta tus nqi ntawm cov primers, lawv yuav sai sai.

Qhov no yog qhov tseem ceeb vim tias hom kev tshawb nrhiav no feem ntau muaj teeb meem hauv lub ntiaj teb tiag tiag, thiab qhov purity ntawm tus qauv, qhov concentration ntawm DNA, lossis muaj lwm yam DNA yuav tsis zoo.

Tsis tas li ntawd, lub hom phiaj ntawm tus lej luam yuav txawv ntawm ntau yam, thiab cov tshuaj reagents, cov khoom siv yas, lossis cov cuab yeej siv yuav txawv ntawm cov uas siv thaum teeb tsa kev sim.

P6| Qhov kub thiab txias gradient qhia qhov sib txawv robustness ntawm PCR nrhiav pom.

A. Siv Bioline's Sensifast SYBR mastermix (catalog number BIO-98050) los ua PCR ntawm cDNA npaj los ntawm tib neeg lub hlwb RNA.

B. Siv Bio-Rad's CFX qPCR ntsuas ntsuas los sau daim ntawv qhia kev nthuav dav thiab kev sib cais nkhaus ntawm apalene (NM_033207, F: GCCATGGAAGGAAAGTGACAGACC, R: CTCATGTGTGGGTGAATCTCCTAGG).

C. Amplification graph thiab melting nkhaus ntawm ACSBG1 (NM_015162.4, F: CTACACTTCCGGCACCACTGG, R: GTCCACGTGATATTGTCTTGACTCAG).

D. Amplification graph and dissolution curve of GFAP (NM_002055.5, F: TGGAGAGGAAGATTGAGTCGCTGG, R: CGAACCTCCTCCTCGTGGAATCTTC).

E. Cqs kaw ntawm qhov sib txawv annealing kub, qhia qhov sib txawv ntawm Cq kaw nyob rau hauv ib tug 7C kub gradient.

P 6 qhia txog qhov tshwm sim ntawm qhov kev sim tsis zoo, qhov twg qPCR tau ua los ntawm kev siv gradient Tas ntawm 59C thiab 67C (P 6a), siv primers rau peb tib neeg lub hlwb-cov noob caj noob ces.

Nws tuaj yeem pom los ntawm cov duab nthuav qhia tias Opalin primers nyob deb ntawm qhov zoo tagnrho vim tias lawv qhov kev pom zoo Ta ntau yog nqaim heev (Daim duab 6b), uas yog, Cqs tau nthuav dav, uas ua rau Cqs tau zoo piv rau lawv qhov kev pom Cqs qis.

Txoj kev tshawb pom no tsis ruaj khov thiab tuaj yeem ua rau muaj qhov ua kom zoo tshaj plaws.Yog li ntawd, cov khub ntawm primers yuav tsum tau redesigned.Tsis tas li ntawd, qhov kev ntsuam xyuas nkhaus melting (inset) qhia tau hais tias qhov tshwj xeeb ntawm txoj kev tshawb pom no kuj tseem muaj teeb meem, vim tias qhov nkhaus melting ntawm txhua Ta yog txawv.

ACSBG1 txoj kev tshawb nrhiav pom nyob rau hauv P 6c yog qhov muaj zog dua li Opalin nrhiav kom tau txoj hauv kev saum toj no, tab sis nws tseem nyob deb ntawm qhov zoo tshaj plaws, thiab nws zoo li yuav txhim kho.

Txawm li cas los xij, peb hais txog tias tsis muaj qhov tsim nyog kev sib txuas ntawm qhov muaj zog thiab qhov tshwj xeeb, vim tias qhov kev sib cais nkhaus tsim los ntawm txoj kev tshawb pom no qhia tau hais tias tus nqi siab tshaj plaws hauv tag nrho Tas (inset).

Ntawm qhov tod tes, qhov kev ntsuam xyuas kev ua tau zoo yog qhov ua tau ntev dua, ua rau Cqs zoo sib xws hauv ntau yam Tas, xws li hauv GFAP qhov kev sim qhia hauv P 6d.

Qhov sib txawv ntawm Cqs tau txais nyob rau hauv tib 8 degrees Celsius ntau dua yog tsawg dua 1, thiab qhov kev sib cais nkhaus (inset) lees paub cov yam ntxwv ntawm qhov ntsuas kub no.Nws yog tsim nyog sau cia tias qhov suav Tas thiab qhov tseeb Ta ntau yuav txawv heev.

Muaj ntau cov txheej txheem tsim los pab cov kws tshawb fawb tsim cov primers zoo, feem ntau yog raws li cov kev cai ntev-tsim thiab ntau cov xim tau them rau 3′ kawg ntawm cov primers.Nws feem ntau pom zoo kom suav nrog G lossis C ntawm 3' kawg thiab ob lub G lossis C hauv paus (GC clamp), tab sis tsis pub ntau tshaj ob ntawm 5 lub hauv paus kawg.

Hauv kev xyaum, cov kev cai no tuaj yeem coj cov kws tshawb fawb, tab sis lawv tsis tas yuav raug rau txhua qhov xwm txheej.

P7 |3' kawg ntawm cov primer muaj me ntsis cuam tshuam rau qhov tshwj xeeb lossis kev ua haujlwm zoo.

A. Txoj hauj lwm ntawm cov primers rau tib neeg HIF-1α (NM_181054.2) noob.

B. Siv Agilent Brilliant III SYBR Ntsuab niam cawv (Cat. No. 600882) los nthuav rau 6 yam khoom xeem.

C. Amplification graph thiab melting nkhaus sau los ntawm Bio-Rad's CFX qPCR ntsuas thiab 3'end primers.NTCs qhia hauv liab.

D. Cqs cov ntaub ntawv ntawm txhua yam kev xeem

Piv txwv li, qhov tshwm sim hauv P 7 cuam tshuam txoj cai 3′ kawg.Tag nrho cov qauv tsim tsim ib qho txiaj ntsig zoo ib yam, nrog tsuas yog ob qho kev sib xyaw ua ke ua rau tsis muaj kev nthuav dav hauv NTC.

Txawm li cas los xij, peb tsis tuaj yeem txhawb nqa cov txiaj ntsig ntawm GC clip, vim tias qhov no, siv A lossis T raws li qhov siab tshaj plaws ntawm 30 lub hauv paus tsis txo qhov tshwj xeeb.

Kuaj C, qhov twg F primer xaus rau hauv GGCC, tau sau Cqs hauv NTCs, qhia tias ib tus yuav xav zam cov kab ke ntawm 30-kawg.Peb hais ntxiv tias tib txoj hauv kev los txiav txim siab qhov zoo tshaj plaws 3'k qhov kawg ntawm ib khub primer yog los ntsuas qee tus neeg sib tw primers sim.

Amplification efficiency

Qhov tseem ceeb, txawm hais tias tsis yog qhov tshwj xeeb PCR tshawb pom tsis tuaj yeem dhau los ua qhov tshwj xeeb, qhov ua kom pom tseeb tuaj yeem hloov kho thiab ua kom tiav ntau txoj hauv kev los ntawm kev hloov cov enzyme, niam cawv, tshuaj ntxiv, thiab kev caij tsheb kauj vab.

Txhawm rau ntsuas qhov ua tau zoo ntawm PCR kev tshawb pom, nws yog qhov zoo tshaj plaws los siv cov txheej txheem dilution ntawm 10 lossis 5 npaug ntawm lub hom phiaj nucleic acid, uas yog, "tus qauv nkhaus txoj kev".

Yog tias PCR amplicons lossis cov hom phiaj DNA hluavtaws tau siv los tsim cov qauv nkhaus, cov kev sib txuas ntawm cov hom phiaj no yuav tsum tau muab sib xyaw nrog cov DNA keeb kwm yav dhau los (xws li genomic DNA).

P8 |Dilution nkhaus los ntsuas qhov ua tau zoo ntawm PCR.

A. Siv primers rau HIF-1: F: AAGAACTTTTAGGCCGCTCA thiab R: TGTCCTGTGGTGACTTGTCC thiab Agilent's Brilliant III SYBR Green mastermix (catalog naj npawb 600882) rau PCR thiab melting nkhaus tej yam kev mob.

B. 100 ng RNA tau thim rov qab, diluted 2 zaug, thiab serially diluted cDNA kuaj tau diluted 5 zaug rau 1 ng human genomic DNA.Lub melting nkhaus yog qhia nyob rau hauv lub inset.

C. Cov tshuaj tiv thaiv RT, dilution, thiab serial dilution tau rov ua dua rau tus qauv cDNA thib ob, thiab cov txiaj ntsig tau zoo sib xws.

P 8 qhia ob tus qauv nkhaus, siv tib txoj kev tshawb pom ntawm ob qhov sib txawv cDNA cov qauv, qhov tshwm sim yog tib yam kev ua haujlwm, kwv yees li 100%, thiab tus nqi R2 kuj zoo ib yam, uas yog, qhov degree ntawm qhov haum ntawm cov ntaub ntawv sim thiab cov kab regression los yog cov ntaub ntawv degree ntawm linearity.

Ob qho qauv nkhaus yog sib piv, tab sis tsis zoo ib yam.Yog tias lub hom phiaj yog txhawm rau txheeb xyuas qhov tseeb ntawm lub hom phiaj, nws yuav tsum tau muab sau tseg tias nws tsis tuaj yeem muab cov ntawv luam ntawm tus lej suav yam tsis tau piav qhia qhov tsis meej.

P9 |Kev ntsuas qhov tsis paub tseeb cuam tshuam nrog kev suav nrog siv tus qauv nkhaus.

A. Siv primers rau GAPDH (NM_002046) ua PCR thiab melting nkhaus tej yam kev mob.F: ACAGTTGCCATGTAGACC thiab R: TAACTGGTTGAGCACAGG thiab Bioline's Sensifast SYBR mastermix (catalog naj npawb BIO-98050).

B. Amplification daim ntawv, melting nkhaus thiab txheem nkhaus kaw nrog Bio-Rad's CFX qPCR ntsuas.

C. Standard nkhaus graph thiab 95% kev ntseeg siab lub sij hawm (CI).

D. Tus lej luam thiab 95% kev ntseeg siab ncua sij hawm ntawm peb Cq qhov tseem ceeb tau los ntawm qhov nkhaus dilution.

P 9 qhia tau hais tias rau qhov kev ntsuam xyuas zoo, qhov sib txawv ntawm ib qho qauv nkhaus yog kwv yees li 2 zaug (95% kev ntseeg siab ib ntus, yam tsawg kawg nkaus rau qhov siab tshaj), uas tej zaum yuav yog qhov sib txawv me me uas tuaj yeem xav tau.

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