Quantitative Gene Expression Analysis in Cotton

Total RNAs were isolated from the indicated plant tissues using the RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics-rich) (Tiangen, DP411, China). The first-strand cDNAs were synthesized from 1 ug total RNAs using the HiScript® III RT SuperMix Kit for qPCR (+ gDNA wiper) (Vazyme, R323-01, China). qRT-PCR was performed on the LightCycler® 480 system (Roche, Switzerland) using the ChamQ Universal SYBR qPCR Marster Mix Kit (Vazyme, Q711, China). GhUBQ7 (GenBank accession No.DQ116441) was used as the internal references, and the relative expression levels of GhPRC2 genes were calculated using the 2^−ΔΔCT method. The gene specific primer sequences were designed on the qPCR Primer database (https://biodb.swu.edu.cn/qprimerdb/) [74 (link)], and listed in Additional file 7: Table S5. In each biological replicate, both the At and Dt-derived primer pairs were used. And at least three biological repeats were performed.