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

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