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.
Protocols
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Mapping Transcription Initiation Sites with Primer Extension
(Laboratory of Dr. Bart Frank, Oklahoma Medical Research Foundation)
The primer extension reaction is used to determine the start site(s) of RNA transcription for a particular gene. One uses a radiolabelled primer that is complementary to a region towards the 5' end of the transcript, and reverse transcribes a single stranded DNA molecule towards the 5' end of the RNA. The size of the labelled single-stranded DNA is then determined on a sequencing gel relative to an M13 ladder.
http://www.omrf.org/omrf/research/09/FrankLab/Prim...
Added: Sat Jan 31 2009, Hits: 3292, Reviews: 0 Write review Cached -
Pol II in vitro Transcription and Primer Extension Assay
(The Hahn Lab, The Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute, Seattle)
http://labs.fhcrc.org/hahn/Methods/biochem_meth/po...
Added: Tue May 14 2002, Hits: 1720, Reviews: 0 Write review Cached -
Primer Extension
(Promega)
http://www.promega.com/tbs/tb113/Tb113.html
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Primer Extension
(Bowtell Lab)
Including procedures for primer labeling and primer extension assay.
http://grimwade.biochem.unimelb.edu.au/bowtell/mol...
Added: Tue May 14 2002, Hits: 638, Reviews: 0 Write review Cached -
Primer Extension
(Genomics Facility, University of Edinburgh)
Protocol gives procedures for primer labeling, reverse transcription from radioactive primer, purification by phenol/chloroform extraction, and running the PAGE gel.
http://staff.biology.ed.ac.uk/services/equipment/g...
Added: Sat Jan 31 2009, Hits: 2219, Reviews: 0 Write review Cached -
Primer Extension of Total Yeast RNA
(The Hahn Lab, The Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute, Seattle)
http://labs.fhcrc.org/hahn/Methods/mol_bio_meth/pr...
Added: Tue May 14 2002, Hits: 1179, Reviews: 0 Write review Cached -
Primer Extention Assay
(Morimoto Lab, Northwestern University)
http://www.biochem.northwestern.edu/morimoto/resea...
Added: Tue Mar 03 2009, Hits: 2993, Reviews: 0 Write review -
Primer Labeling for Primer Extension Assay and Primer Extension
(Bowtell Lab Manual, Peter MacCallum Cancer Institute)
http://www.pmci.unimelb.edu.au/core_facilities/man...
Added: Tue May 14 2002, Hits: 460, Reviews: 0 Write review Cached