TY - JOUR
T1 - Validation of reference genes for quantitative measurement of immune gene expression in shrimp
AU - Dhar, Arun K.
AU - Bowers, Robert M.
AU - Licon, Kate S.
AU - Veazey, Gregory
AU - Read, Betsy
PY - 2009/5
Y1 - 2009/5
N2 - To accurately measure the relative expression of a target gene, mRNA expression data is routinely normalized with reference to an internal control gene. We examined the transcriptional stability of four internal control genes, β-actin, glyceroldehyde-3 phosphate dehydrogenase (GAPDH), elongation factor1-alpha (EF1-α), and 18S ribosomal RNA (18S rRNA) while measuring the mRNA expression of a gene encoding a pattern recognition protein, lipopolysaccharide and glucan binding protein (LGBP) gene, in healthy and white spot syndrome virus (WSSV) infected shrimp (Penaeus stylirostris) before and after (4, 8, 16 and 32 h) challenge using real-time RT-PCR. Here, we describe a method to rank the internal control genes based on a linear regression analysis. This analysis enables us to analyze the multivariate data set, e.g. time course study samples with control and treatment groups. Using the linear regression analysis and the WSSV-challenged time course samples, GAPDH was found to be the most stable internal control gene followed by the genes EF1-α, 18S rRNA and β-actin. Using the program geNorm, GAPDH was also found to be the most stable gene followed by the genes EF1-α, β-actin and 18S rRNA. Using the program NormFinder, the ranking of the internal control genes were in the order of EF1-α > GAPDH > 18S rRNA > β-actin. The ability to compare the healthy and WSSV infected samples in parallel by the regression analysis makes this method a very useful approach while identifying the optimal reference gene for gene expression analysis.
AB - To accurately measure the relative expression of a target gene, mRNA expression data is routinely normalized with reference to an internal control gene. We examined the transcriptional stability of four internal control genes, β-actin, glyceroldehyde-3 phosphate dehydrogenase (GAPDH), elongation factor1-alpha (EF1-α), and 18S ribosomal RNA (18S rRNA) while measuring the mRNA expression of a gene encoding a pattern recognition protein, lipopolysaccharide and glucan binding protein (LGBP) gene, in healthy and white spot syndrome virus (WSSV) infected shrimp (Penaeus stylirostris) before and after (4, 8, 16 and 32 h) challenge using real-time RT-PCR. Here, we describe a method to rank the internal control genes based on a linear regression analysis. This analysis enables us to analyze the multivariate data set, e.g. time course study samples with control and treatment groups. Using the linear regression analysis and the WSSV-challenged time course samples, GAPDH was found to be the most stable internal control gene followed by the genes EF1-α, 18S rRNA and β-actin. Using the program geNorm, GAPDH was also found to be the most stable gene followed by the genes EF1-α, β-actin and 18S rRNA. Using the program NormFinder, the ranking of the internal control genes were in the order of EF1-α > GAPDH > 18S rRNA > β-actin. The ability to compare the healthy and WSSV infected samples in parallel by the regression analysis makes this method a very useful approach while identifying the optimal reference gene for gene expression analysis.
KW - Immune gene
KW - LGBP
KW - Real-time RT-PCR
KW - Reference gene
KW - SYBR Green
KW - Shrimp
UR - http://www.scopus.com/inward/record.url?scp=64149115872&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=64149115872&partnerID=8YFLogxK
U2 - 10.1016/j.molimm.2009.02.020
DO - 10.1016/j.molimm.2009.02.020
M3 - Article
C2 - 19297025
AN - SCOPUS:64149115872
SN - 0161-5890
VL - 46
SP - 1688
EP - 1695
JO - Molecular Immunology
JF - Molecular Immunology
IS - 8-9
ER -