caa a22 4500 445319100 CHVBK 20180317142657.0 cr unu---uuuuu 170323e20110401xx s 000 0 eng 10.1007/s10126-010-9277-z doi (NATIONALLICENCE)springer-10.1007/s10126-010-9277-z 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 [Elektronische Daten] [Christian De Santis, Carolyn Smith-Keune, Dean Jerry] 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 4weeks 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 4weeks. 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. Springer Science+Business Media, LLC, 2010 Quantitative real-time RT-PCR nationallicence Reference genes nationallicence Fasting nationallicence Lates calcarifer nationallicence RT-qPCR normalization methods nationallicence De Santis Christian Aquaculture Genetics Research Program, School of Marine and Tropical Biology, James Cook University, 4811, Townsville, QLD, Australia aut Smith-Keune Carolyn Aquaculture Genetics Research Program, School of Marine and Tropical Biology, James Cook University, 4811, Townsville, QLD, Australia aut Jerry Dean Aquaculture Genetics Research Program, School of Marine and Tropical Biology, James Cook University, 4811, Townsville, QLD, Australia aut Marine Biotechnology Springer-Verlag 13/2(2011-04-01), 170-180 1436-2228 13:2<170 2011 13 10126 https://doi.org/10.1007/s10126-010-9277-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10126-010-9277-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- De Santis Christian Aquaculture Genetics Research Program, School of Marine and Tropical Biology, James Cook University, 4811, Townsville, QLD, Australia aut NATIONALLICENCE 700 1- Smith-Keune Carolyn Aquaculture Genetics Research Program, School of Marine and Tropical Biology, James Cook University, 4811, Townsville, QLD, Australia aut NATIONALLICENCE 700 1- Jerry Dean Aquaculture Genetics Research Program, School of Marine and Tropical Biology, James Cook University, 4811, Townsville, QLD, Australia aut NATIONALLICENCE 773 0- Marine Biotechnology Springer-Verlag 13/2(2011-04-01), 170-180 1436-2228 13:2<170 2011 13 10126 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer