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一種新穎簡便的熒光實時RT
最近更新時間：2016-4-14
提 供 商：上海士鋒生物科技有限公司資料大?。?/span>48.3KB
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一種新穎簡便的熒光實時 RT-PCR相對定量方法的建立
A Novel and Convenient Relative quantitative Method of Fluorescence Real Time RT-PCR Assay Based on Slope of Standard Curve
稿件編號：2005-0257
中文關鍵詞：熒光實時定量 RT-PCR ，相對定量，定量，標準曲線， mRNA 定量，斜率
英文關鍵詞：fluorescence real-time quantitative RT-PCR, relative quantification, absolute quantification, standard curve, mRNA quantification, slope
基金項目：江蘇省高校自然科學指導計劃資助項目（04KJD180044）和江蘇大學人才科研啟動基金資助項目（2281270002）.
作者單位
張馳宇 江蘇大學醫(yī)學技術學院，鎮(zhèn)江 212001
徐順高 江蘇大學醫(yī)學技術學院，鎮(zhèn)江 212001
黃新祥 江蘇大學醫(yī)學技術學院，鎮(zhèn)江 212001
中文摘要：
為建立一種新穎、簡便的熒光實時 RT-PCR 相對定量方法，根據實時定量標準曲線，推導出相對定量基因表達的公式 . 公式顯示相對表達指數只與 CT 值和標準曲線的斜率相關 . 構建標準曲線的標準品需要通過克隆和體外轉錄獲得，實驗過程繁瑣 . 當人為成比例增減標準品各個稀釋度的具體拷貝數時，標準曲線的斜率并不改變，說明標準曲線斜率與標準品的具體拷貝數無關 . 因此，新的相對定量方法可以用任何一個待測樣品的總 RNA ( 或 cDNA) ，經系列稀釋后作為標準品，來構建相對定量標準曲線，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果 ( 差異小于4%) ，而傳統(tǒng)的 2 -ΔΔCT法卻表現出較大的定量誤差 . 這些結果表明，新的相對定量方法是一種簡便、準確和的定量基因表達的方法 .
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.