PJB-2022-133
Selecting the appropriate qPCR reference genes to explore melatonin function in Highland barley
Xiangyu Wei
Abstract
Ten candidate reference genes (ACT, EF-1α, E2, GAPDH, HSP90, PGK, PKABA1, SAMDC, TUBα, and TUBβ6) was analyzed by quantitative polymerase chain reaction (qPCR) to select the most stable reference genes in Highland barley (Hordeum vulgare L. var. nudum. Hook. f.) seedlings after treatment with various doses of melatonin. Then, we evaluated their expression stability using four tools (geNorm, NormFinder, BestKeeper and RefFinder). The results indicated that EF-1α was the most stable reference gene under all treatment conditions. No significant difference was detected between the temporal expression profiles of the well-studied clock gene GI, after calibration by EF-1α alone as the optimal reference gene, or after calibration by double references, such as EF-1α and other optimized references. These results confirm that selecting the most appropriate reference genes could guarantee the verification and accuracy of target gene expression analysis by qPCR in Highland barley, which will facilitate further studies on the function of melatonin in Highland barley.
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