Zenilman M E, Graham W, Tanner K, Shuldiner A R
Department of Surgery, Johns Hopkins University, Hopkins Bayview Medical Center, Baltimore, Maryland 21224.
Anal Biochem. 1995 Jan 1;224(1):339-46. doi: 10.1006/abio.1995.1049.
Advances in our understanding of molecular and cellular physiology necessitate that mRNA levels for specific growth factors and other rare transcripts be measured quantitatively in small samples. Conventional methods such as Northern blot analysis and solution hybridization/ribonuclease protection are not sufficiently sensitive. We now report the theory, development, and validation of a rapid and highly sensitive assay, the RNA/DNA quantitative polymerase chain reaction (RD-PCR), which uses a competitive PCR approach to measure the number of copies of a specific mRNA per cell. Total nucleic acid (RNA and genomic DNA) is isolated from cells in culture. The mRNA of interest is first reverse-transcribed with an oligomer bearing a complementary sequence specific for the mRNA at its 3'-end, and a sequence complementary to an intron of the desired gene at the 5'-end. Competitive PCR is then performed in the presence of the cDNA product and endogenous genomic DNA, with an upstream primer complementary to the exon sequence of the gene of interest, and a downstream primer complementary to the intron sequence that was tagged to the cDNA. The cell's own genomic DNA is thereby used as the internal standard. To control for the efficiency of reverse transcription, a standard curve is used in each assay. The technique was validated by comparing the quantitation of insulin-like growth factor I (IGF-I) mRNA in two human cell lines by RD-PCR and by RNase protection analysis. Both methods gave similar numbers of copies of IGF-I mRNA per cell. For accurate analysis, RNase protection required at least 10(7) cells; RD-PCR required as little as 10(2) cells.(ABSTRACT TRUNCATED AT 250 WORDS)
随着我们对分子和细胞生理学认识的不断深入，有必要对小样本中特定生长因子和其他稀有转录本的mRNA水平进行定量测定。诸如Northern印迹分析和溶液杂交/核糖核酸酶保护等传统方法灵敏度不足。我们现在报告一种快速且高度灵敏的检测方法——RNA/DNA定量聚合酶链反应（RD-PCR）的原理、开发及验证，该方法采用竞争性PCR方法来测定每个细胞中特定mRNA的拷贝数。从培养的细胞中分离出总核酸（RNA和基因组DNA）。首先，用一种寡聚物对感兴趣的mRNA进行逆转录，该寡聚物在其3'端带有与mRNA特异互补的序列，在5'端带有与所需基因的一个内含子互补的序列。然后，在cDNA产物和内源性基因组DNA存在的情况下进行竞争性PCR，使用与感兴趣基因的外显子序列互补的上游引物，以及与标记到cDNA上的内含子序列互补的下游引物。细胞自身的基因组DNA因此用作内标。为控制逆转录效率，每次检测都使用标准曲线。通过比较RD-PCR和核糖核酸酶保护分析对两种人类细胞系中胰岛素样生长因子I（IGF-I）mRNA的定量，验证了该技术。两种方法得出的每个细胞中IGF-I mRNA的拷贝数相似。为进行准确分析，核糖核酸酶保护至少需要10^7个细胞；RD-PCR仅需低至10^2个细胞。（摘要截短于250字）
