Selection of stable reference genes for quantitative real-time PCR in porcine gastrointestinal tissues

Abstract

The selection of stable reference genes is an essential aspect of controlling for the many technical variables that influence the final outcome in quantitative real-time PCR (qPCR). In this study, we evaluated the suitability of 8 commonly used reference genes based on their efficiency and dynamic range in porcine gastrointestinal tissues. The genes included ACTB, B2M, GAPDH, HMBS, HPRT, PPIA, 18S rRNA, and YWHAZ. We then sub-selected four reference genes ( ACTB, B2M, GAPDH, PPIA) of ‘medium’ abundance, and evaluated their stability using the geNorm algorithm across three unchallenged and immunologically challenged (bacterial lipopolysaccharide (LPS) and concanavalin A (ConA)) tissue types (liver, ileum, colon) of porcine origin. Normalisation factors were generated for each group using the most stable reference pairs for each group. The most stable reference pairs based on their M values are summarised as follows: 1) liver ( B2M, GAPDH), 2) ileum ( B2M, PPIA), 3) colon ( B2M, PPIA), 4) all treatment groups ( B2M, GAPDH), and 5) entire data set ( B2M, GAPDH). ACTB was the least stable gene in all groups. The stability of selected reference genes for the ileum and colon differed from the liver but was not significantly altered by immunological challenges with LPS and ConA.