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一種新穎簡(jiǎn)便的熒光實(shí)時(shí)RT
最近更新時(shí)間：2016-4-14
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一種新穎簡(jiǎn)便的熒光實(shí)時(shí) RT-PCR相對(duì)定量方法的建立
A Novel and Convenient Relative quantitative Method of Fluorescence Real Time RT-PCR Assay Based on Slope of Standard Curve
稿件編號(hào)：2005-0257
中文關(guān)鍵詞：熒光實(shí)時(shí)定量 RT-PCR ，相對(duì)定量，定量，標(biāo)準(zhǔn)曲線， mRNA 定量，斜率
英文關(guān)鍵詞：fluorescence real-time quantitative RT-PCR, relative quantification, absolute quantification, standard curve, mRNA quantification, slope
基金項(xiàng)目：江蘇省高校自然科學(xué)指導(dǎo)計(jì)劃資助項(xiàng)目（04KJD180044）和江蘇大學(xué)人才科研啟動(dòng)基金資助項(xiàng)目（2281270002）.
作者單位
張馳宇 江蘇大學(xué)醫(yī)學(xué)技術(shù)學(xué)院，鎮(zhèn)江 212001
徐順高 江蘇大學(xué)醫(yī)學(xué)技術(shù)學(xué)院，鎮(zhèn)江 212001
黃新祥 江蘇大學(xué)醫(yī)學(xué)技術(shù)學(xué)院，鎮(zhèn)江 212001
中文摘要：
為建立一種新穎、簡(jiǎn)便的熒光實(shí)時(shí) RT-PCR 相對(duì)定量方法，根據(jù)實(shí)時(shí)定量標(biāo)準(zhǔn)曲線，推導(dǎo)出相對(duì)定量基因表達(dá)的公式 . 公式顯示相對(duì)表達(dá)指數(shù)只與 CT 值和標(biāo)準(zhǔn)曲線的斜率相關(guān) . 構(gòu)建標(biāo)準(zhǔn)曲線的標(biāo)準(zhǔn)品需要通過克隆和體外轉(zhuǎn)錄獲得，實(shí)驗(yàn)過程繁瑣 . 當(dāng)人為成比例增減標(biāo)準(zhǔn)品各個(gè)稀釋度的具體拷貝數(shù)時(shí)，標(biāo)準(zhǔn)曲線的斜率并不改變，說明標(biāo)準(zhǔn)曲線斜率與標(biāo)準(zhǔn)品的具體拷貝數(shù)無關(guān) . 因此，新的相對(duì)定量方法可以用任何一個(gè)待測(cè)樣品的總 RNA ( 或 cDNA) ，經(jīng)系列稀釋后作為標(biāo)準(zhǔn)品，來構(gòu)建相對(duì)定量標(biāo)準(zhǔn)曲線，獲得斜率 . 與定量法比較，新方法獲得了基本相同的斜率和非常一致的定量結(jié)果 ( 差異小于4%) ，而傳統(tǒng)的 2 -ΔΔCT法卻表現(xiàn)出較大的定量誤差 . 這些結(jié)果表明，新的相對(duì)定量方法是一種簡(jiǎn)便、準(zhǔn)確和的定量基因表達(dá)的方法 .
英文摘要：
In order to develop a novel, simple and effective relative quantitative method using fluorescence real-time RT-PCR, the formulas were derived from the standard curve for quantifying relative expression of mRNA. The formulas show that the relative expression index is only associated with CT value and the slope of standard curve. RNA (DNA) standard for absolute quantitative standard curve is obtained generally by cloning and transcription in vitro. When input copy numbers of serial diluted RNA standards were increased or decreased n-fold (n > 0) proportionally, the slope of standard curve remains invariable, suggesting that the slope is independent of actual copy numbers of RNA standard. Therefore, anyone of tested RNA (cDNA) samples could be used after serial dilution as RNA standards to obtain slope. Compared with absolute quantitative method, the present one appears to have a more excellent consistency (difference less than 4%) in the relative expression indices with absolute quantitative method, than traditional relative method 2-ΔΔCT (difference more than 5%). The results show that the method described here is simple, precise and cost-effective for relative quantifying gene expression.