Identification of Stable Reference Genes for Normalization of Gene Expression In Avian 25+iel-nk Cells Following Newcastle Disease Virus Infection

The use of multiple reference genes has long been advocated as a robust method for achieving accurate normalization in quantitative reverse transcription polymerase chain reaction (RT-qPCR). These internal reference genes are employed to normalize target gene expression and are expected to remain invariant regardless of treatment conditions, such as viral infection. However, several studies have reported instability in the expression of commonly used reference genes, including beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18s rRNA, under various experimental conditions. This study aimed to identify suitable reference genes for normalising genes associated with chicken intraepithelial lymphocyte natural killer (IEL-NK) cells following infection with different strains of Newcastle Disease Virus (NDV). Three commonly used reference genes were selected and two IEL-NK cell-related genes, B-NK and NK-lysin, for normalisation purposes. The mean M and V values for the combinations of reference genes (18S/ACTB; 18S/GAPDH; ACTB/GAPDH; and 18S/ACTB/GAPDH) were calculated using the Target Stability Value program in CFX Manager software version 3.1. Based on the recommended stability values, the combination of ACTB and GAPDH demonstrated the most stable expression among the reference gene combinations, while the inclusion of 18S resulted in unstable expression. Therefore, combining ACTB and GAPDH is recommended as suitable reference genes for normalising chicken IEL-NK cell-related genes following NDV infection.