NDLI: 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

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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

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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

Content Provider	Springer Nature Link
Author	Santis, Christian Smith Keune, Carolyn Jerry, Dean R.
Copyright Year	2010
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.
Starting Page	170
Ending Page	180
Page Count	11
File Format	PDF
ISSN	14362228
Journal	Marine Biotechnology
Volume Number	13
Issue Number	2
e-ISSN	14362236
Language	English
Publisher	Springer-Verlag
Publisher Date	2010-03-23
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Quantitative real-time RT-PCR Reference genes Fasting Lates calcarifer RT-qPCR normalization methods Microbiology Freshwater & Marine Ecology Zoology Engineering
Content Type	Text
Resource Type	Article
Subject	Aquatic Science Biotechnology

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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