Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample.
Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample.
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DOI：
10.1251/bpo20
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发表时间：
2001-11-16
影响因子：
6.4
通讯作者：
Scambia G
中科院分区：
文献类型：
--
作者：
Marone M;Mozzetti S;De Ritis D;Pierelli L;Scambia G
我们描述了在我们的实验室中优化的半定量 RT-PCR 方案，可从少至 10,000 个细胞中提取 RNA，并测量每个样本中多个目标 mRNA 的表达水平。该程序在人红白血病细胞系 TF-1 上进行了优化，但已成功用于原代细胞和不同的细胞系。我们描述了 Bcl-2 水平分析的详细程序。醛缩酶 A 用作内部对照，以标准化样品间总 RNA 量的变化和反应效率。对于所有定量技术，在所有优化步骤中都必须非常小心：此处描述了确保粗略定量（半定量）分析的必要控制，以及来自 TF-1 细胞中 TGF-β1 影响的研究的示例。
We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-β1 in TF-1 cells.