Competitive reverse transcription-polymerase chain reaction for quantifying murine AIDS virus
| Autor: | K. Akarid, I. Hatin, Martine Sinet, B. Desforges |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Gene Expression Regulation
Viral DNA Complementary viruses Molecular Sequence Data Gene Products gag Biology Virus Replication Polymerase Chain Reaction Defective virus Virus Mice Murine Acquired Immunodeficiency Syndrome Virology Gene expression Murine leukemia virus Animals RNA Messenger Cloning Molecular Cells Cultured Defective Viruses biology.organism_classification Molecular biology Reverse transcriptase Actins Reverse transcription polymerase chain reaction Leukemia Virus Murine Mice Inbred C57BL Real-time polymerase chain reaction Helper virus RNA Viral Electrophoresis Polyacrylamide Gel Female Spleen Plasmids |
| Zdroj: | Journal of virological methods. 62(2) |
| ISSN: | 0166-0934 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect