Ab geneamp pcr system 9700

To validate RNA-seq results, the gene expression from nine randomly selected DEGs was analyzed using a two-step real-time quantitative PCR (qRT-PCR). Two independent biological and three technical replicates were performed. First, 1 μg total RNA per sample was reverse-transcribed into first-strand cDNA using the M-MuLV Reverse Transcriptase kit (Fermentas, Burlington, ON, Canada), following manufacturer protocol. After 10× dilution, cDNA was used as templates for qRT-PCR (AB GeneAmp PCR System 9700;Applied Biosystems, Foster City, CA, USA). The reaction mixture was prepared using the FastStart Universal SYBR Green Master (ROX) kit (ROCHE) following manufacturer protocol and added to an optical 384-well plate. The jute ELF gene was selected as the endogenous control [28 ]. Primers for the DEGs and ELF are listed in Supplementary File S1. Relative expression levels were determined using the comparative Ct method [29 (link)].

To validate the results of the high-throughput sequencing, qRT-PCR of the same samples used for transcriptome sequencing was performed in an AB GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The qRT-PCR was carried out in a two-step procedure according to the method by reported by Yangs⁵³ . The thermal cycle consisted of an initial denaturation at 95 °C for 10 min followed by 40 cycles at 95 °C for 10 s and 58 °C for 30 s. The relative expression levels were analysed according to a protocol described by Livak and Schmittgen^{54 (link)}. Eight DEGs randomly selected from the RNA-seq results were used for validation; the jute ELF gene was selected as the endogenous control⁵⁵ . Each PCR reaction was conducted in triplicates. The primer sequence of DEGs and ELF gene are listed in Supplementary File 4.