Doris P A, Oefner P J, Chilton B S, Hayward-Lester A
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
J Chromatogr A. 1998 May 8;806(1):47-60. doi: 10.1016/s0021-9673(97)00514-1.
We have analyzed the utility of ion-pair reversed-phase HPLC for gene quantification by competitive reverse transcriptase polymerase chain reaction (RT-PCR). Competitive RT-PCR reactions employed various RNA competitors which shared high sequence similarity to the native transcripts for which they served as references. Competitive reactions resulted in the detection of two reaction products when reactions were analyzed by agarose gel electrophoresis, but three products when analyzed by HPLC. The third product was demonstrated to be a heteroduplex formed between mixed strands of native and competitor amplicons. Mathematical analysis of these competitive reactions indicated that identification and quantification of the heteroduplexes were essential to produce accurate gene quantification. PCR amplification efficiency was shown to be identical for native and competitor transcripts. However, RT efficiency differences were observed which may be sequence dependent. These differences were highly consistent across reactions for the same native and competitor inputs. Increasing the sequence similarity resulted in a competitor which had the same RT efficiency as the native transcript. Titration of various levels of competitor against native RNA resulted in the expected linear relationships which had slopes of unity. Quantitation could be performed with similar precision in single tube comparisons in which the initial abundance of the native transcript was calculated by knowledge of the final reaction product ratio and the initial competitor input level. The assay system is highly accurate, i.e. the measured level of gene expression reflected the actual copy number of the gene present in the sample. This was demonstrated by performing reactions in which known amounts of native transcript were quantified and the amount estimated by the assay was shown to be the same as the known amount added to the reaction. A similar approach has been devised for examining the relative levels of alternatively spliced isoforms. In this system, primers were selected to produce reaction products which served as their own internal competitors (by spanning the alternative splice site). Hormonal dependence of the ratio of abundance of two isoforms of the rabbit RUSH-1 gene was demonstrated.
我们分析了离子对反相高效液相色谱法在通过竞争性逆转录聚合酶链反应（RT-PCR）进行基因定量中的应用。竞争性RT-PCR反应采用了各种RNA竞争物，这些竞争物与它们作为参考的天然转录本具有高度的序列相似性。当通过琼脂糖凝胶电泳分析反应时，竞争性反应产生了两种反应产物，但通过高效液相色谱分析时则产生了三种产物。第三种产物被证明是天然扩增子和竞争物扩增子的混合链之间形成的异源双链体。对这些竞争性反应的数学分析表明，异源双链体的鉴定和定量对于产生准确的基因定量至关重要。结果表明，天然转录本和竞争物转录本的PCR扩增效率相同。然而，观察到逆转录效率存在差异，这可能与序列有关。对于相同的天然和竞争物输入，这些差异在各反应中高度一致。增加序列相似性会产生与天然转录本具有相同逆转录效率的竞争物。用不同水平的竞争物滴定天然RNA会产生预期的线性关系，其斜率为1。在单管比较中可以以类似的精度进行定量，其中通过最终反应产物比率和初始竞争物输入水平来计算天然转录本的初始丰度。该检测系统高度准确，即所测量的基因表达水平反映了样品中存在的基因的实际拷贝数。通过进行已知量的天然转录本定量的反应来证明这一点，并且检测所估计的量与添加到反应中的已知量相同。已经设计了一种类似的方法来检查可变剪接异构体的相对水平。在该系统中，选择引物以产生作为其自身内部竞争物的反应产物（通过跨越可变剪接位点）。证明了兔RUSH-1基因两种异构体丰度比率的激素依赖性。
