Lear W, McDonnel M, Kashyap S, Boer P H
University of Ottawa Heart Institute, Ontario, Canada.
Biotechniques. 1995 Jan;18(1):78-80, 82-3.
The ability to accurately measure mRNA levels in samples of total RNA is essential for studies on control of gene expression. The mRNAs from the housekeeping gene for phosphoglycerate kinase (PGK-1) can serve as a quality control for RNA samples. We describe an enzyme-linked immunosorbent assay (ELISA) method for mRNA determination by Q-RT-PCR, a quantitative reverse transcriptase-mediated PCR assay with competitive internal standards. After PCR, two biotinylated capture primers, one specific for PGK-1 cDNA and another one for internal standard, are annealed in separate assays so that each can attach DNA to a streptavidin-coated microplate. The captured DNA is either internally labeled with digoxigenin (DIG) or is "developed" after annealing with DIG-labeled primers. Bound DNA is then quantitated by adding DIG-specific antibody with attached alkaline phosphatase and measuring phosphatase activity with a chromogenic substrate and a plate reader. We compared different capturing methods and various primers labeled with DIG at their 3' ends. We determined that amplified PGK-1 DNA specifically captured with biotinylated primers was efficiently assayed with random p(dN)6-DIG.
准确测量总RNA样本中mRNA水平的能力对于基因表达调控研究至关重要。磷酸甘油酸激酶（PGK-1）看家基因的mRNA可作为RNA样本的质量控制指标。我们描述了一种通过Q-RT-PCR（一种带有竞争性内标的定量逆转录酶介导的PCR检测法）测定mRNA的酶联免疫吸附测定（ELISA）方法。PCR后，两条生物素化的捕获引物，一条针对PGK-1 cDNA，另一条针对内标，在单独的检测中退火，以便每条引物都能将DNA附着到链霉亲和素包被的微孔板上。捕获的DNA要么用洋地黄毒苷（DIG）进行内部标记，要么在用DIG标记的引物退火后“显色”。然后通过加入附着有碱性磷酸酶的DIG特异性抗体并用显色底物和酶标仪测量磷酸酶活性来定量结合的DNA。我们比较了不同的捕获方法以及在其3'端用DIG标记的各种引物。我们确定用生物素化引物特异性捕获的扩增PGK-1 DNA能有效地用随机p(dN)6-DIG进行检测。
