Tea tree miRNA fluorescent quantitative PCR reference gene under low temperature stress as well as screening method and application of reference gene
A fluorescence quantitative, internal reference gene technology, applied in the field of molecular biology, can solve problems such as unstable expression
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[0044] Selection of miRNAqRT-PCR internal reference candidate genes and primer design
[0045] Six internal reference genes commonly used in plant miRNAqRT-PCR (U6, 18SrRNA, 26SrRNA, 5.8SrRNA, 5SrRNA and GAPDH) and three tea tree miRNAs with relatively stable expression (cs-miR396b, Pc-222-3p and Pc-45545- 5p), using these 9 miRNAs as internal reference candidate genes. The sequences of miRNAU6, 18SrRNA, 26SrRNA, 5.8SrRNA, 5SrRNA and GAPDH were obtained from NCBI, and the sequences of tea tree miRNAcs-miR396b, Pc-222-3p and Pc-45545-5p were obtained from tea tree genome sequencing data and miRNA low-temperature sequencing library. The nucleotide sequences are respectively shown in SEQ ID NO: 3, SEQ ID NO: 1 and SEQ ID NO: 4.
[0046] Primer Premier 5.0 software was used to design upstream and downstream primers of internal reference genes. The upstream and downstream primers of U6, 18SrRNA, 26SrRNA, 5.8SrRNA, 5SrRNA and GAPDH are designed according to the specific sequence, ...
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