Quantitation of TNFa mRNA in bronchoalveolar lavage cells by polymerase chain reaction using cRNA internal standard

TNFα mRNA in human bronchoalveolar lavage(BAL)cells was quantitated by a olymerase chain reaction(PCR)using a cRNA internal standard,cRNA molecules were in vitro transcribed from pAW108 plasmid in which the sequences of upstream primer and omolementary sequences of downstream primer of TNFα were inserted.The cRNA and mRNA extracted from BAL cells were mixed and reverse-transcribed into cDNA.The resultant cDNA mixture was 1:3 diluted and amplified by PCR using TNFα specific primers.~(32)p-labeled upstream primers were included in the PCR reaction,cDNA fragments amplified was run on a 3% agarose gel.The radioactivity of positive bands was determined in a scintillation ounter.After plotting variable template concentrations of the internal standard pAW108 cRNA and the number of BAL cells against the radioactivity of their PCR products,the levels of TNFα mRNA in BAL cells were quantitated by comparision to those of cRNA internal standard.