Total RNAs were extracted using the CTAB method (Chang et al., 1993 ) with slight modification. cDNAs were generated using HiScript II Q RT SuperMix with gDNA wiper (Vazyme). For miR397, the stem‐loop primer for reverse transcription was added during cDNA synthesis (Chen et al., 2005 (link)). PcActin and PcU6 served as reference genes for analysis of expression levels of mRNA and miR397, respectively. The U6 sequence of pear was obtained by local BLAST search against the P. bretschneideri genome (http://peargenome.njau.edu.cn ) using U6 of maize (GenBank accession number X52315.1). RT‐qPCR was carried out on a CFX96 Real‐time System (Bio‐Rad) using ChamQ Universal SYBR qPCR Master Mix (Vazyme). Each analysis included three biological replicates for each treatment. Relative expression levels were quantified by the 2−ΔΔCt method (Livak & Schmittgen, 2001 (link)). Primers used in RT‐qPCR are listed in Table S6 .
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Quantification of mRNA and miRNA Expression
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Corresponding Organization : Huazhong Agricultural University
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Variable analysis
independent variables
- None explicitly mentioned
- Expression levels of mRNA and miR397
- PcActin and PcU6 served as reference genes for analysis of expression levels of mRNA and miR397, respectively
- The U6 sequence of pear was obtained by local BLAST search against the P. bretschneideri genome
- Positive control: PcActin and PcU6 served as reference genes
- Negative control: None explicitly mentioned
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