Strategy of the use of 28S rRNA as a housekeeping gene in real-time quantitative PCR analysis of gene transcription in insect cells infected by viruses.

Quantitative real-time reverse transcription-PCR (qRT-PCR) has been used widely to measure gene transcription regulation in cells. qRT-PCR must include one or more internal housekeeping genes to normalize data collection. A strategy to use the host cell 28S rRNA as a housekeeping gene in qRT-PCR analysis of gene transcription of insect cells infected by baculovirus and ascovirus was developed. It has been found that the 28S rRNA reverse primer can be incorporated in the oligo-dT-primed cDNA synthesis reaction. In such a way, amplification of 28S cDNA showed lower and less variable cycle thresholds in cells infected by viruses than by using only oligo-dT and other published housekeeping genes such as the TATA box binding protein (TBP) gene, the peptidyl prolyl isomerase A (PPI) gene and the ribosomal protein 13 (L13) gene. Incorporation of the 28S reverse primer in oligo-dT-primed cDNA synthesis also does not interfere with the detection of other polymerase II transcribed genes.