Turbo dna free kit

Determination of related gene as following method: For quantitative real-time polymerase chain reaction (RT-PCR) analysis, total RNA was extracted using an RNAiso Plus kit (Takara, Beijing, China). A TURBO DNA-free kit (Sigma–Aldrich, Shanghai, China) was used to remove genomic DNA. Single-stranded cDNA was synthesized using a PrimeScriptH RT kit (Takara). The SYBR green method (Takara) was performed on a CFX96 Touch real-time PCR detection system. The relative expression levels of the amplified products were calculated using the 2^−ΔΔCt method. Primers for the selected unigenes were designed online using NCBI Primer-BLAST (Bio-Rad, Hercules, CA, USA). CsActin (HQ420251.1) was used as the internal housekeeping gene (Table 1). The primer design was according to the previous study [24 (link),25 (link)].

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).

In this study, the Trizol Method (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from the grape leaves of the control group and salt stressed groups. A TURBO DNA-free kit (Sigma-Aldrich, St. Louis, MO, USA) was used to extract genomic DNA from the total RNA. The quality and concentration levels of extracted total RNA were measured using an ultraviolet spectrophotometer (Implen, Germany), and used for RNA-Seq and real-time quantitative PCR. In addition, all of the treatments implemented in this study contained three biological replicates.