Abstract
The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies.
Highlights
The use of gene expression analysis intends to clarify biological processes involved with several conditions in living organisms enabling the identification of diagnostic markers as therapeutic targets in the treatment of diseases [1]
The gene RPL5 was classified in the sixth position with the software NormFinder (Table 2) differing from the Bestkeeper and GeNorm results
The gene HMBS had a divergent classification among BestKeeper, GeNorm and NormFinder, ranking this gene, respectively, in the third, sixth and eighth positions (Table 2)
Summary
The use of gene expression analysis intends to clarify biological processes involved with several conditions in living organisms enabling the identification of diagnostic markers as therapeutic targets in the treatment of diseases [1]. The quantitative PCR (qPCR) is a fast, easy-to-use technique that provides simultaneous measurement of gene expression in many different samples for a limited number of genes [2]. In qPCR, fluorescent dyes are used to combine the amplification and detection steps of the PCR reaction in a single tube [3,4]. QPCR has been widely used for validating RNA-seq results due to its high sensitivity and precision [5]. When comparing to other techniques, it has advantages such as sensitivity, real-time detection.
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