Selection of suitable internal controls for gene expression normalization in rats with spinal cord injury

Abstract

There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research. In this study, we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes: Actb, Ankrd27, CypA, Gapdh, Hprt1, Mrpl10, Pgk1, Rictor, Rn18s, Tbp, Ubc, Ubxn11, and Ywhaz. Our results show that the expression of Ankrd27, Ubc, and Tbp were stable after spinal cord injury, while Actb was the most unstable internal control gene. Ankrd27, Ubc, Tbp, and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression. Target gene expression levels and changes over time were similar when Ankrd27, Ubc, and Tbp were used as internal controls but different when Actb was used as an internal control. We recommend that Ankrd27, Ubc, and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research. This study was approved by the Administration Committee of Experimental Animals, Jiangsu Province, China (approval No. 20180304-008) on March 4, 2018.

Keywords

• genorm analysis; reference genes; internal control genes; normalization; normfinder analysis; reverse transcription-quantitative polymerase chain reaction; spinal cord injury; stability of gene expression