RANDOM PRIMER P(DN)(6)-DIGOXIGENIN LABELING FOR QUANTITATION OF MESSENGER-RNA BY Q-RT-PCR AND ELISA

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 a s a quality control for RNA samples. We describe an enzyme-linked immu nosorbent assay (ELISA) method for mRNA determination by Q-RT-PCR, a q uantitative reverse transcriprase-mediated PCR assay, with competitive internal standards. After PCR, two biotinylated capture printers, one specific for PGK-1 cDNA and another one for internal standard, are an nealed in separate assays so that each can attach DNA to a streptacidi n-coated microplate. The captured DNA is either internally labeled wit h digoxigenin (DIG) or is ''developed'' after annealing with DIG-label ed primers. Bound DNA is then quantitated by adding DIG-specific antib ody with attached alkaline phosphatase and measuring phosphatase activ ity with a chromogenic substrate and a plate reader. We compared diffe rent capturing methods and various primers labeled with DIG at their 3 ' ends. We determined that amplified PGK-1 DNA specifically captured w ith biotinylated primers was efficiently assayed with random p(dN)(6)- DIG.