Selection of the appropriate reference genes for relative quantitative reverse transcription polymerase chain reaction (qRT-PCR) in canine pulmonary arteries

Abstract

A normalisation of targeted genes using the appropriate reference genes is necessary for reliable quantitative
reverse transcription polymerase chain reaction (qRT-PCR) experiments. This study aimed to investigate the
appropriate reference genes and the numbers of the genes required for qRT-PCR in canine pulmonary arteries.
Pulmonary arteries were collected from twenty-three dogs at necropsy. Candidate reference genes including ribosomal
protein L32 (RPL32), ribosomal protein L13a (RPL13A), ribosomal protein S18 (RPS18), TATA box binding protein
(TBP), beta-2-microglobulin (B2M), ribosomal protein S5 (RPS5), ribosomal protein S19 (RPS19) and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) from pulmonary arteries were examined by qRT-PCR and analysed using four
major algorithms, including geNorm, NormFinder, the comparative delta-CT method, and RefFinder. The optimal
number of genes was analysed using geNorm. The ranking of the appropriate reference genes from all algorithms was
evaluated by RefFinder. The appropriate reference genes for the normalisation of the targeted gene in canine
pulmonary arteries are RPS19 and RPL32.