Measurement of mRNA by solution hybridization with 32P-labelled single stranded cRNA probe ("SP6 test"). Comparison with a 32P-labelled single stranded cDNA as hybridization probe ("S1 test") for measurement of AVP mRNA.
Radioactively labelled cRNA for the rat AVP gene exon C was synthetized from a pSP64-vector and used for solution hybridization measurement of AVP mRNA ("SP6 test"). For comparison hybridization was carried out with a gel-purified radioactively labelled cDNA probe synthetized by primer extension of AVP gene exon C cloned into an M13mp9 phage vector DNA ("S1 test"). Both tests had a comparable sensitivity of up to 0.2 pg AVP mRNA. Under optimal hybridization conditions kinetics were similar in both tests. The fast and easy preparation of large amounts of labelled cRNA probe and simple determination of absolute amounts of mRNA by saturation kinetics without need of a mRNA standard makes the "SP6 test" an attractive alternative to the known "S1 test". The "SP6 test" should be applicable for a wide variety of genes.
D000818:Animals; D004247:DNA / Q000235:genetics*; D007700:Kinetics; D008297:Male; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D012313:RNA / Q000235:genetics*; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic; D014667:Vasopressins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D010761:Phosphorus Radioisotopes; D018075:RNA, Complementary; D012333:RNA, Messenger; D014667:Vasopressins; D012313:RNA; D004247:DNA