Random primer p(dN)6-digoxigenin labeling for quantitation of mRNA 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 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.
Lear
W
W
University of Ottawa Heart Institute, Ontario, Canada.
McDonnel
M
M
Kashyap
S
S
Boer
P H
PH
D001483:Base Sequence; D017931:DNA Primers; D004076:Digoxigenin / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*
D017931:DNA Primers; D012333:RNA, Messenger; D010735:Phosphoglycerate Kinase; D004076:Digoxigenin