The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue

Catríona M Dowling; Dara Walsh; John C Coffey; Patrick A Kiely

doi:10.12688/f1000research.7656.2

The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue

F1000Res. 2016 Jan 22:5:99. doi: 10.12688/f1000research.7656.2. eCollection 2016.

Authors

Catríona M Dowling¹, Dara Walsh², John C Coffey², Patrick A Kiely¹

Affiliations

¹ Department of Life Sciences, and Materials and Surface Science Institute, University of Limerick, Limerick, Ireland; Health Research Institute, University of Limerick, Limerick, Ireland; Graduate Entry Medical School, University of Limerick, Limerick, Ireland.
² 4i Centre for Interventions in Infection, Inflammation and Immunity, Graduate Entry Medical School, University of Limerick, Limerick, Ireland.

Abstract

Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) remains the most sensitive technique for nucleic acid quantification. Its popularity is reflected in the remarkable number of publications reporting RT-qPCR data. Careful normalisation within RT-qPCR studies is imperative to ensure accurate quantification of mRNA levels. This is commonly achieved through the use of reference genes as an internal control to normalise the mRNA levels between different samples. The selection of appropriate reference genes can be a challenge as transcript levels vary with physiology, pathology and development, making the information within the transcriptome flexible and variable. In this study, we examined the variation in expression of a panel of nine candidate reference genes in HCT116 and HT29 2-dimensional and 3-dimensional cultures, as well as in normal and cancerous colon tissue. Using normfinder we identified the top three most stable genes for all conditions. Further to this we compared the change in expression of a selection of PKC coding genes when the data was normalised to one reference gene and three reference genes. Here we demonstrated that there is a variation in the fold changes obtained dependent on the number of reference genes used. As well as this, we highlight important considerations namely; assay efficiency tests, inhibition tests and RNA assessment which should also be implemented into all RT-qPCR studies. All this data combined demonstrates the need for careful experimental design in RT-qPCR studies to help eliminate false interpretation and reporting of results.

Keywords: Colon Cancer; NormFinder; Normalisation; Quantitative real-time PCR; Reference Genes.

Grants and funding

This work was supported by grants received from the Irish Cancer Society Grant CRS12DOW (to CD), the Mid-Western Cancer Foundation and funding from Science Foundation Ireland grant 13/CDA/2228 (to PK).