Validating Tea Transcriptome Unigenes via qRT-PCR

To validate the accuracy of unigenes obtained from the assembled tea transcriptome datasets and profiling of gene expression via RNA-Seq, qRT-PCR was performed for the selected unigenes. Total RNA was isolated from tea buds using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, Shanghai, China). RNA samples were treated by the TURBO DNA-free™ Kit (Sigma-Aldrich, Shanghai, China) to remove traces of genomic DNA. Single-stranded cDNAs used for qRT-PCR were synthesized using a Prime-Script™ Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). qRT-PCR was carried out using the SYBR green method for detection of double-stranded PCR products (TaKaRa, Dalian, China). An IQ5 real-time PCR detection system (Bio-Rad) was utilized in this study as previously described [62 (link)]. The tea β-actin gene was used as an internal reference gene (HQ420251.1, https://www.ncbi.nlm.nih.gov/nuccore/HQ420251.1) [69 (link)]. The primers for 17 selected unigene in this study were designed by Primer Premier 5.0 software (PREMIER Biosoft Company, http://www.premierbiosoft.com/index.html, Additional file 7).