Clinical Microbiology: Open Access

Verifying the stability of selected genes for normalization in qPCR experiments of Spodoptera frugiperda cells during AcMNPV infection

3^rd International Conference on Clinical Microbiology & Microbial Genomics

September 24-26, 2014 Valencia Convention Centre, Spain

Salem T Z, Allam W R and Thiem S M

Posters: Clin Microbial

Abstract:

It is challenging to find stably expressed genes that can be used as reference genes for qRT-PCR during viral infection, in order to obtain accurate gene expression data. Autographa californica nucleopolyhedro virus (AcMNPV) is known to globally shut off transcription in Sf21 cells and to modify their cytoskeletons. During a previous comprehensive microarray study, ecdysoneless (ECD) gene was found to be stable during the course of AcMNPV infection to Sf21 cells. In this study, seven host genes were selected for validation as references for gene expression experiments using the quantitative real time polymerase chain reaction (qRT-PCR). Three of them, ECD, myosin, and ribosomal protein L35 (L35) genes, showed stability in our previous microarray study, the others, actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S ribosome (28S), are commonly employed as reference genes for qRT-PCR. In AcMNPV-infected Sf21 cells, qRT-PCR showed that ECD and 28S were the most stable transcripts throughout the time course of infection, which was terminated at 48 hours post infection (hpi). As expected gene expression of cytoskeleton genes such as actin, tubulin, and myosin were unstable, but declined dramatically as the infection progress. GAPDH, a commonly used reference gene, expression also declined over time. Although L35 gene expression appeared stable by microarray data, it was not stable when qRT-PCR was used. These results suggest ECD is a reliable reference gene for qRT-PCR experiments during AcMNPV infection. The choice of 28S as a stable reference gene should come second as its abundance may hinder establishing a correct baseline value for data analysis.

Biography :

Salem T Z has obtained his PhD in Microbiology and Cell Science from the University of Florida. He was a research associate at a number of universities including University of Florida, Miami University, and Michigan State University, USA. He is Associate Professor of biology in Biomedical Sciences at University of Science and Technology, Zewail city of Science and Technology, Egypt. He has published more than 18 papers in reputed journals most of them in the virology field.