Competitive reverse transcription-polymerase chain reaction for quantifying murine AIDS virus.
The causative agent of murine AIDS (MAIDS) is the defective murine leukemia virus BM5d, that requires the replication-competent ecotropic MuLV (BM5e) helper virus. We developed a competitive quantitative PCR method including specific internal standards to quantify the expression of BM5d in the spleen of infected mice and to characterize BM5d expression kinetics following experimental infection. Specimen RNA was reverse-transcribed and co-amplified with a competitive template containing a gag sequence specific for BM5d that can be discriminated from that corresponding to wild-type cDNA by the presence of a unique restriction site, Bg/II. PCR products were quantified by means of densitometric analysis after ethidium bromide staining of gels. To standardise the RNA extraction and reverse transcription steps, the amount of defective-virus mRNA was compared to a constant copy number of murine beta actin mRNA. LP-BM5 production was measured in the spleen of infected mice. Defective gag mRNA production was compared to that of the ecotropic virus. The mRNA level of the defective virus and the titre of replicative virus increased with the duration of infection, and the amount of defective virus mRNA correlated with the titre of replicating virus.
Desforges
B
B
INSERM U13, Hôpital Bichat-Claude Bernard, Paris, France.
Hatin
I
I
Akarid
K
K
Sinet
M
M
D000199:Actins / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015967:Gene Expression Regulation, Viral; D015683:Gene Products, gag / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016183:Murine Acquired Immunodeficiency Syndrome / Q000235:genetics* / Q000821:virology*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000235:genetics; D013154:Spleen / Q000821:virology; D014779:Virus Replication / Q000235:genetics
D000199:Actins; D018076:DNA, Complementary; D015683:Gene Products, gag; D012333:RNA, Messenger; D012367:RNA, Viral