Quantitation of TNFa mRNA in bronchoalveolar lavage cells by polymerase chain reaction using cRNA internal standard
- VernacularTitle:以cRNA为内在参照物测定人支气管肺泡洗出液细胞TNF? mRNA的含量
- Author:
Yongli YU
;
Guizhen YANG
- Publication Type:Journal Article
- Keywords:
Quantitation of mRNA;
Polymerase chain reaction;
Tumor necrosis factor
- From:
Chinese Journal of Immunology
1985;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
TNF? 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 TNF? 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 TNF? 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 TNF? mRNA in BAL cells were quantitated by comparision to those of cRNA internal standard.
