Identification of differentially expressed 5'-end mRNA variants by an improved RACE technique (PEETA).
A rapid and efficient procedure is described for mapping and cloning the 5'-ends of mRNAs, including those generated from a unique gene by alternative splicing and promoter usage. This method involves reverse transcription of the targeted mRNAs from a long, highly labeled specific primer, resolution of the extension products on a DNA sequencing gel, elution and poly(dC) tailing of the single-stranded cDNAs of interest, amplification of these cDNAs by PCR using an oligo(dG) adapter-primer and a gene-specific primer and finally DNA sequencing of the subcloned PCR fragments. The overall method is called PEETA (primer extension, electrophoresis, elution, tailing and amplification).
Flouriot
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EMBL, Meyerhofstrasse 1, D-69117, Heidelberg, Germany.
Brand
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Gannon
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F
D000293:Adolescent; D000328:Adult; D017398:Alternative Splicing; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary; D047628:Estrogen Receptor alpha; D005260:Female; D005821:Genetic Techniques; D014644:Genetic Variation; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D011960:Receptors, Estrogen / Q000235:genetics*
D018076:DNA, Complementary; D047628:Estrogen Receptor alpha; D012333:RNA, Messenger; D011960:Receptors, Estrogen