Top : Molecular Biology : RNA : Primer Extension

Summary: Primer extension is method used to measure the amount of a specific RNA transcript or to map the transcription start (5 end) of a transcript. A primer extension experiment involves the following steps: 1) Total RNA or poly-A RNA is isolated and used as the template; 2) An synthetic oligonucleotide, 20-40 nt in length and complementary to the 5 most end of the know transcript, is end-labeled usually with an isotope; 3) the labeled primer is hybridized to the RNA and then extended using reverse transcriptase to produce single-stranded cDNA using the RNA as the template; 4) The resulting cDNA is subsequently analyzed on a denaturing polyacrylamide gel (PAGE). A manual sequencing reaction is also run along with the primer extension product to help determine the size of the extended products. The length of the cDNA reflects the number of bases between the labeled nucleotide of the primer and 5′-end of the RNA, and the quantity of cDNA product is proportional to the amount of input target RNA.