少量的DNA 序列的準確定量曾經(jīng)是一件很難很累的事情，不過real timePCR 的出現(xiàn)把它變?yōu)楹推胀≒CR 差不多一樣容易。原理是利用能特異標記PCR產(chǎn)物的熒光物質(zhì)，顯示PCR 產(chǎn)物的動態(tài)累積，得到S 型的擴增曲線；假設該曲線的前期PCR 符合指數(shù)性擴增，于是在單純指數(shù)方程的基礎上，通過比較產(chǎn)物積累的速度（時間）來間接比較（進而確定）初始模板的分子數(shù)。我們試驗了標準曲線的制作，以及對免疫相關的目的基因IL-4 轉(zhuǎn)錄水平的定量，而且用HPRT持家基因?qū)L-4 進行歸一化。結(jié)果與RT-PCR 相近。靈敏度，誤差降低，可重復性有望更上一層樓。

關鍵詞：
real time PCR 定量 熒光SYBR 擴增曲線 標準曲線 cDNA

Abstract:
It had been difficult and tiring to quantitate a small amount of DNA until the invention of real time PCR technique, which is as easy as regular PCR.It produces and displays the kinetic accumulation of PCR product (oramplicon)----called amplification curve. Assuming that amplicons are amplified exponentially during the earlier cycles, we make use of the simple equation y=axto compare the start copy number of a gene in different samples, by measuring the threshold cycle (Ct). And with some standard, copy number of a gene can be
quantified. In this experiment we tried making some standard curves. Then we quantified IL-4 cDNA in 3 subsets of T helper cell, normalized by HPRT housekeeping GENE. The result correlates with RT-PCR. However, sensitivity,deviation and precision still need to be improved.

Key word:
real time PCR quantitation fluorescence SYBR Green I amplification curve standard curve cDNA