Normalizing RT-qPCR data: are we getting the right answers? An appraisal of normalization approaches and internal reference genes from a case study in the Finfish Lates calcarifer

De Santis, Christian, Smith-Keune, Carolyn, and Jerry, Dean R. (2011) Normalizing RT-qPCR data: are we getting the right answers? An appraisal of normalization approaches and internal reference genes from a case study in the Finfish Lates calcarifer. Marine Biotechnology, 13 (2). pp. 170-180.

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Abstract

Commonly used normalization approaches for quantitative reverse transcription polymerase chain reaction analyses include (a) input nucleic acids standardization (ΔC q method), (b) normalizing target gene transcript abundance against a single internal reference gene (ΔΔC q method), and (c) geometric averaging of multiple reference gene abundance using the geNorm software. We compared these three approaches to examine expression of a negative muscle growth regulator gene, myostatin-I (mstn-I), in the finfish Lates calcarifer, following 4 weeks of nutritional fasting. Interestingly, these three different approaches led to widely divergent data interpretations. When ΔC q and subsequently ΔΔC q with α-tub as the reference gene were applied to mstn-I expression data, an ∼threefold upregulation of this gene was observed in fasted compared to fed fish. In contrast, mstn-I appeared unchanged when data was normalized against the geometric average of the two apparently most “stable” reference genes (elongation factor-1 α (ef1-α) and rpl8) selected from a panel comprising seven commonly utilized candidate reference genes (18S, cat-D, ef1-α, rpl8, gapdh, ubq, and α-tub). The geNorm software erroneously suggested that ef1-α, rpl8, and ubq were the most “stable” reference genes, whereas ΔC q analysis revealed these genes simply to exhibit similar patterns of regulation in response to fasting. The ΔC q approach showed that α-tub was the least variable in its expression level between fasted and fed fish after 4 weeks. The present study also highlights the importance of validating internal references for each time point under investigation when applying ΔΔC q and suggests that the more cost-effective ΔC q normalization approach, if carefully applied, may in fact produce the most biologically valid results.

Item ID: 16666
Item Type: Article (Research - C1)
ISSN: 1436-2236
Keywords: quantitative real-time RT-PCR; reference genes; fasting; Lates calcarifer, RT-qPCR normalization methods
Date Deposited: 15 May 2011 09:40
FoR Codes: 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060405 Gene Expression (incl Microarray and other genome-wide approaches) @ 50%
07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070401 Aquaculture @ 50%
SEO Codes: 83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830102 Aquaculture Fin Fish (excl. Tuna) @ 100%
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