A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards
A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards
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DOI：
10.1006/mcpr.2000.0288
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发表时间：
2000-04-01
影响因子：
3.3
通讯作者：
Yung, WKA
中科院分区：
文献类型：
--
作者：
Ke, LD;Chen, Z;Yung, WKA
基因表达的定量测量需要一致且可靠的标准。目前至少有两类标准品（内源性和外源性）用于定量 PCR。然而，这两种方法的可靠性尚未经过仔细比较。我们假设可靠的定量 PCR 检测能够检测给定单链 (ss-) cDNA 的已知稀释度。通过测量两种原始 ss-cDNA 制剂的 10 倍和 100 倍稀释样品中的 VEGF ss-cDNA 拷贝数或 GAPDH/VEGF 信号比，测试外源重组 DNA 标准品（VEGF 模拟质粒）和内源表达的 GAPDH 标准品检测稀释因子的能力。使用重组DNA标准品，在原始CaSki细胞ss-cDNAI的10倍和100倍稀释样品中检测到稀释因子分别为10.3和135.0。来自 U-251 MG 细胞的 10 倍和 100 倍稀释 ss-cDNA 中检测到的稀释因子分别为 12.3 和 226.2。另一方面，使用内源 GAPDH 标准品，在原始 U-251 MG 细胞 ss-cDNA 的相同 10 倍和 100 倍稀释液中检测到稀释因子为 2.7 和 8.0。使用相同的内源 GAPDH 标准品，在原始 CaSki 细胞 ss-cDNA 的 10 倍和 100 倍稀释液中检测到的稀释因子均为 4.8。还发现GAPDH mRNA的内源拷贝数比VEGF高约1000倍。 GAPDH 与 VEGF 拷贝数的高内部锁定比以及需要额外的引物对，使得使用丰富的内源标准品在定量或半定量 PCR 中成为不可靠的选择。相比之下，基于外源标准的定量 PCR 被证明是一种准确可靠的基因表达定量方法。 (C) 2000 年学术出版社。
The quantitative measurement of gene expression requires consistent and reliable standards. At least two categories of standards, endogenous and exogenous, are currently used for quantitative PCR. The reliability of these two methods, however, has not been carefully compared. We hypothesized that a reliable quantitative PCR assay would be able to detect known dilutions of a given single-stranded (ss-) cDNA. By measuring VEGF ss-cDNA copy numbers or signal ratios of GAPDH/VEGF in 10 x and 100 x diluted samples of two original ss-cDNA preparations, an exogenous recombinant DNA standard (a VEGF-mimic plasmid) and an endogenously expressed GAPDH standard were tested for their ability to detect dilution factors. Using the recombinant DNA standard, the dilution factor was detected as 10.3 and 135.0 in 10 x and 100 x diluted samples of the original CaSki cell ss-cDNAI respectively. The detected dilution factors were 12.3 and 226.2, respectively, in 10 x and 100 x diluted ss-cDNA from U-251 MG cells. On the other hand, with the endogenous GAPDH standard, the dilution factors were detected as 2.7 and 8.0 in the same 10 x and 100 x dilutions of the original U-251 MG cell ss-cDNA. Using the same endogenous GAPDH standard, the detected dilution factors were both 4.8 in 10 x and 100 x dilutions of the original CaSki cell ss-cDNA. It was also found that the number of endogenous copies of GAPDH mRNA was about 1000 times higher than VEGF. The high internal lockup ratio of GAPDH vs VEGF copy numbers and the requirement for additional primer pairs make the use of an abundant endogenous standard an unreliable choice in quantitative or semi-quantitative PCR. In contrast, exogenous standard-based quantitative PCR was shown to be an accurate and reliable method for the quantitation of gene expression. (C) 2000 Academic Press.