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lixiaojing1318
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熒光定量需要3個(gè)重復(fù)樣本嗎 已有8人參與
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熒光定量需要3個(gè)重復(fù)樣本嗎,還是只要一個(gè)樣品,做3個(gè)重復(fù)? 提完RNA后,需要定量到相同濃度嗎? |
分子生化實(shí)驗(yàn)經(jīng)驗(yàn)積累 | 【分子生物】EPI帖 |
金蟲 (正式寫手)
木蟲 (正式寫手)
金蟲 (正式寫手)
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你說的是biological replicate 和 technical replicate,一般每個(gè)樣本做三到四個(gè)技術(shù)重復(fù),這個(gè)貌似是熒光定量必需的 生物學(xué)重復(fù)就是可以獨(dú)立重復(fù)提取兩三次,分別進(jìn)行實(shí)驗(yàn),熒光定量最好需要生物學(xué)重復(fù)~~ 另外RNA量最好定在一個(gè)范圍里面,雖然有內(nèi)參基因校正,但是大部分綜述推薦,這樣好調(diào)整模版量,并且由于是擴(kuò)增反應(yīng),在2ΔΔct如果擴(kuò)增效率不平行,很可能誤差會(huì)放大,因此ΔΔct也最好都做個(gè)標(biāo)準(zhǔn)曲線,樣品濃度最好在標(biāo)準(zhǔn)曲線范圍內(nèi)。 摘自google~ A technical replicate involves the multiple labeling or reciprocal labeling of the same RNA sample. The purpose of a technical replicate is to control for technical variability within an experiment (array to array variation, reagent variation, dye incorporation, etc.) A well run experiment should have minimal variation thus minimizing the need for technical replicates. A biological replicate involves isolating RNA independently from replicate sources (multiple cell lines, multiple biopsies, multiple patients, etc). The purpose of a biological replicate is to control for biological diversity. Biological replicates are often more telling, and for this reason are “better” than technical replicates, however, biological replicates are often more difficult to obtain [ Last edited by shaquila on 2011-7-19 at 13:47 ] |
銀蟲 (小有名氣)
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