Tripure reagent total rna extraction reagent

Genomic DNA was extracted from three biological replicates of each frozen asexual single root line using the cetyltrimethylammonium bromide (CTAB) plant DNA extraction method. PCR amplification of the specific fragment was then performed using it as a template to verify the successful transfer of the overexpressing fragment to screen for overexpressing-positive hairy root asexual root lines. Total RNA was isolated from three biological replicates of each overexpressing-positive hairy root asexual root line by using the TRIpure Reagent Total RNA Extraction Reagent from BioTeke, Beijing, China. and reverse-transcribed into cDNA using the HiFiScript gDNA Removal cDNA Synthesis Kit from CWBIO, Taizhou, China. The transcript level of the target transcript in each sample was measured by qPCR in ABI 7500 Fast Real-Time PCR System with Actin 1 as the internal reference gene [35 (link)] using UltraSYBR Mixture (Low ROX) from Jiangsu Cowin Biotech Co., Taizhou, China (Tables S2 and S4). Three technical replicates were performed for each of these samples.

TruSeq Stranded Total RNA with Ribo-Zero Gold (Illumina, United States); AgencourtAMPure XP (BECKMAN COULTER, United States); Agencourt RNAClean XP(BECKMAN COULTER, United States); QubitRNA Assay Kit (Life Technologies, United States); QubitdsDNA Assay Kit (Life Technologies, United States); Bioanalyzer 2100 RNA-6000 Nano Kit (Aglient, United States); Bioanalyzer 2100 DNA-1000 Kit (Agilent, United States); SuperScript II Reverse Transcriptase (Invitrogen, United States); miRNA first strand cDNA synthesis kit (Accurate Biology, China); TRIpure Reagent Total RNA Extraction Reagent (Bioteke Corporation, China); primer (Tsingke Biotechnology Co., China).

Total RNA of ginseng hairy roots was isolated using the TRIpure Reagent Total RNA Extraction Reagent (Bioteke, Beijing, China). The concentration and quality of the RNA were determined by Scandrop 100 (Analytic Jena AG). cDNA synthesis from the RNA was carried out using the PrimeScript^™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China). The concentration of cDNA was also determined by Scandrop 100 and then diluted to 100 ng/μL for qRT-PCR.