Total RNA was extracted by using Plant RNA Kit (Omega Bio-Tek, Doraville, GA, USA) according to the manufacturer’s instructions. The cDNAs were generated by RT-PCR using the StarScript III RT Mix Kit (GenStar, Beijing, China). The qRT-PCR analyses were performed using a qTower3G Real-time PCR System (Analytik Jena AG, Jena city, Germany) and SYBR® Green Fast Mixture (GenStar, Beijing, China). The ZaActin and NbActin genes were used as internal references to normalize the gene expression levels. The relative gene expression levels were calculated using the 2−ΔΔCt method [45 (link)]. The primers used for real-time PCR are listed in Table S1 .
Sybr green fast mixture
Manufactured by GenStar
Sourced in Germany
SYBR® Green Fast Mixture is a ready-to-use solution for quantitative real-time PCR. It contains SYBR® Green I dye, which binds to double-stranded DNA and emits fluorescence upon binding. The mixture also includes all necessary components for PCR amplification, including DNA polymerase, dNTPs, and buffer.
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2 protocols using sybr green fast mixture
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
Auxin Pathway Transcriptome Analysis
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The auxin pathway of the transcriptome analysis was randomly selected for RT-qPCR verification. RNA was extracted, and cDNA was further prepared from apical buds and stem bases. Gene-specific primers were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) (Supplementary Table S1). RT-qPCR system and procedure were referenced in our previous research4 (link). The Ljubiquitin gene was used as an internal reference to normalize the gene expression levels. The RT-qPCR analyses were performed using a qTower3G Real-time PCR System (Analytik Jena AG, Jena city, Germany) and SYBR® Green Fast Mixture (GenStar, Beijing, China). Normalized transcript abundances were calculated using the 2−ΔΔCT method.
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