Ct kukosha ndiyo inonyanya kukosha mhedzisiro fomu yefluorescent quantitative PCR.Inoshandiswa kuverenga gene expression misiyano kana gene kopi nhamba.Saka chii chinonzi Ct kukosha kwefluorescence quantification inonzi inonzwisisika?Nzira yekuona sei hunobudirira huwandu hweCt kukosha?
Chii chinonzi Ct Value?
Munguva yeqPCR yekusimudzira maitiro, nhamba inoenderana yeamplification cycles (Cycle Threshold) apo chiratidzo chefluorescence chechigadzirwa chakasimudzirwa chasvika pachikumbaridzo chefluorescence.C inomirira Cycle uye T inomirira Threshold.Zvichitaurwa zviri nyore, iyo Ct kukosha ndiyo nhamba yematenderedzwa anoenderana kana yekutanga template amplification inosvika imwe huwandu hwechigadzirwa muqPCR.Izvo zvinonzi "chimwe chinhu chechigadzirwa" chichazotsanangurwa zvakare gare gare.
Ko Ct value inoita sei?
1.Relationship pakati peexponential amplification, template yakawanda uye Ct value
Sezvineiwo, majini muqPCR anounganidzwa neexponential amplification mushure meimwe nhamba yekutenderera.Hukama huri pakati pehuwandu hweamplification cycles uye huwandu hwezvigadzirwa ndeichi: Yakawedzerwa chigadzirwa huwandu = yekutanga template huwandu × (1 + En) nhamba yekutenderera.Nekudaro, iyo qPCR maitiro haisi nguva dzose iri mune yakanaka mamiriro.Kana huwandu hwechigadzirwa chakasimudzirwa chasvika "chimwe chigadzirwa huwandu", huwandu hwekutenderera panguva ino ndiyo Ct kukosha, uye iri muexponential amplification nguva.Ukama huri pakati peCt value uye huwandu hwekutanga template: Pane hukama hwemutsara pakati peCt kukosha kwetemplate uye logarithm yenhamba yekutanga yekopi yetemplate.Iyo yakakwirira yekutanga template concentration, iyo idiki iyo Ct kukosha;iyo yakaderera yekutanga template concentration, iyo yakakura kukosha kweCt.
2.Amplification curve, fluorescence threshold uye imwe PCR chigadzirwa chiyero
Huwandu hweiyo qPCR amplification chigadzirwa inoratidzwa zvakananga muchimiro chefluorescent chiratidzo, kureva, iyo amplification curve.Muchikamu chekutanga chePCR, kukwidziridzwa kuri pasi pemamiriro akanaka, huwandu hwematenderedzwa idiki, kuunganidzwa kwechigadzirwa kudiki, uye mwero wefluorescence haugone kunyatsosiyaniswa kubva kune fluorescence kumashure.Mushure meizvozvo, iyo fluorescence inowedzera uye inopinda muchikamu cheexponential.Huwandu hwePCR chigadzirwa hunogona kuwonekwa pane imwe nguva apo PCR reaction ingori muchikamu cheexponential, chinogona kushandiswa se "chimwe chikamu chechigadzirwa", uye zvekutanga zvemukati zve template zvinogona kutorwa kubva pane izvi.Naizvozvo, iyo fluorescence chiratidzo chesimba inoenderana nehumwe huwandu hwechigadzirwa ndiyo fluorescence chikumbaridzo.
Muchikamu chekupedzisira chePCR, iyo amplification curve haicharatidzi exponential amplification, uye inopinda mumutsara wechikamu uye chikamu cheplateau.
3.Reproducibility yeCt values
Kana iyo PCR kutenderera inosvika kutenderera nhamba yeCt kukosha, ichangopinda muchokwadi exponential amplification nguva.Panguva ino, kukanganisa kuduku hakuna kukwidziridzwa, saka kuberekazve kweCt kukosha kwakanakisisa, ndiko kuti, template imwe chete inowedzerwa panguva dzakasiyana kana mumachubhu akasiyana panguva imwe chete.Amplification, iyo yakawanikwa Ct kukosha inogara iripo.
1.Amplification kunyatsoshanda En
PCR amplification performance inoreva kushanda kunoita iyo polymerase inoshandura jini kuti rikwidziridzwe kuita amplicon.Kubudirira kwekusimudzira kana molecule imwe yeDNA ikashandurwa kuita mamorekuru maviri eDNA i100%.Amplification kunyatsoshanda kunowanzo kuratidzwa seEn.Kuti ufambise kuongororwa kwezvinyorwa zvinotevera, izvo zvinokanganisa kugona kwekusimudzira zvinounzwa muchidimbu.
| Kufurira zvinhu | tsananguro | Kutonga sei? |
| A. PCR inhibitors | 1. The template DNA ine zvinhu zvinodzivisa PCR reaction, zvakadai seprotein kana detergents.2. Iyo cDNA mushure mekunyorwa kwereverse ine huwandu hwepamusoro hwe template RNA kana RT reagent zvikamu, izvo zvinogonawo kuvharidzira zvinotevera PCR reaction. | 1. Kana pane kusvibiswa kunogona kutongwa nekuyera chiyero cheA260 / A280 uye A260 / A230 kana RNA electrophoresis.2. Kana iyo cDNA yakanyungudutswa maererano neimwe reshiyo mushure mekunyorwa kwereverse. |
| B. Zvisizvo primer design | Maprimers haatore zvakanaka | Tarisa maprimer-dimers kana hairpins, mismatches, uye dzimwe nguva inotenderera intronic dhizaini. |
| C. Yakashata PCR reaction program design | 1. Maprimers haakwanise kuverengera zvinobudirira2. Kusakwana kusunungurwa kweDNA polymerase 3. Kureba kwekushisa kwepamusoro kweDNA polymerase basa rakaderera | 1. The annealing tembiricha yakakwira kupfuura kukosha kweTM kweprimer2. Pre-denaturation nguva ipfupi 3. Nguva yedanho rega rega rekuita maitiro yakarebesa |
| D. Kusakwana kusanganisa reagents kana pipetting zvikanganiso | Mune iyo reaction system, iyo yemuno kusangana kwePCR maitiro ezvikamu zvakanyanyisa kana kusaenzana, zvichikonzera kusiri-exponential amplification yePCR amplification. | |
| E. Amplicon Length | Hurefu hweamplicon hwakarebesa, hunodarika 300bp, uye kugona kwekusimudzira kwakadzikira. | Tarisa kuti urefu hweamplicon huri pakati pe80-300bp |
| F. Kufurirwa kweqPCR reagents | Iko kusungirirwa kweDNA polymerase mu reagent kwakadzikira kana kuwanda kwemaion mubuffer hakuna kugadziridzwa, zvichikonzera kuti Taq enzyme basa risasvike pakakwirira. | Kutsunga kweamplification kunyatsoshanda neyakajairwa curve |
2.Range yeCt values
Ct kukosha kubva ku15-35.Kana iyo Ct kukosha iri pasi pe15, inofungidzirwa kuti kukwidziridzwa kuri mukati mechikamu chenguva yekutanga uye chikumbaridzo chefluorescence hachina kusvika.Zvakanaka, pane hukama hwemutsara pakati peCt kukosha uye logarithm yekutanga kopi nhamba ye template, kureva, chiyero chepamusoro.Kuburikidza neyakajairwa curve, kana iyo amplification inobudirira iri 100%, iyo yakaverengerwa Ct kukosha kwehuwandu hweiyo imwe kopi nhamba yejini inenge yakapoteredza 35. Kana yakakura kudarika 35, nhamba yekutanga yekopi ye template inotaura zvishoma kudarika 1, iyo inogona kuonekwa sechinhu chisina maturo.
Kune akasiyana gene Ct mitsara, nekuda kwekusiyana kweiyo gene kopi nhamba uye amplification kunyatsoita muyekutanga template huwandu, zvinodikanwa kugadzira chiyero che curve yejini uye kuverenga mutsara wekutsvaga mutsara wejini.
3.Kufurira zvinhu zveCt kukosha
Kubva pahukama huri pakati pehuwandu hwemazamu ekusimudza uye huwandu hwechigadzirwa: huwandu hwechigadzirwa chakasimudzirwa = huwandu hwekutanga template × (1 + En) nhamba yekutenderera, zvinogona kuonekwa kuti pasi pemamiriro ezvinhu akanaka, chiyero chepakutanga template uye En ichave nemigumisiro yakaipa paCt value inobatwa.Musiyano mumhando yetemplate kana kukwidziridza kunyatsoita kuchaita kuti kukosha kweCt kuve hombe kana kudiki.
4.Ct kukosha kwakanyanya kana kudiki
Nguva yekutumira: Feb-22-2023
