TITLE:
Selection of Reference Genes in Equine White Blood Cells for Real Time PCR Normalization Following Extracorporeal Shock Wave Therapy
AUTHORS:
Zibin Jiang, Jinwen Chen, Cornelius E. Uboh, Mary A. Robinson, Lawrence R. Soma
KEYWORDS:
Reference Genes; Real Time PCR; Normalization; Equine; White Blood Cell; Extracorporeal Shock Wave Therapy
JOURNAL NAME:
American Journal of Molecular Biology,
Vol.4 No.2,
April
1,
2014
ABSTRACT: Selection of
proper reference genes (RGs) is an essential step needed for accurate
normalization of results from genomic studies. Expression of RGs is regulated
by many factors such as species, age, gender, type of tissue, the presence of
disease, and the administration of therapeutic treatment. The aim of the
present study was to identify optimal RGs in a set of blood samples collected
at different time points (0, 24, 48, 72 h) from horses following administration of
extracorporeal shock wave therapy (ESWT). The mRNA expression of twelve RGs: HPRT1, ACTB, HSP90A, SDHA, GUSB, B2M, UBC, NONO, TBP, H6PD, RPL32, GAPDH was determined using real time
quantitative polymerase chain reaction (qPCR). An SAS program developed on the
algorithm of geNorm, SASqPCR, was used to determine stability of the expression
and the number of optimal RGs. The results showed that the range of
quantification cycle (Cq) values of the evaluated genes varied between
17 and 26 cycles, and that one optimal RG, ACTB,
was sufficient for normalization of gene expression. Results of stability of
expression demonstrated that ACTB was
the optimal choice for all the samples studied. Notably, in samples collected
at 72 h post ESWT, TBP showed a significant change in the expression level, and was
not suitable for use as a RG. These results substantiate the importance of
validating and selecting an appropriate RG.