Reversetra ace qpcr rt kit

Using qRT-PCR, the expression patterns of 12 genes (Gene ID: Bra003517, Bra013923, Bra020878, Bra028899, Bra031485, Bra038089, Bra034061, Bra035732, Bra010802, Bra012938, Bra00944, and Bra025833) were analyzed (Primers are listed in Additional file 6: Table S3). Actin was used as the reference gene according to [81 (link)]. In brief, cDNA was synthesized using ReverseTra Ace qPCR-RT Kit (Toyobo, Japan). The reverse transcription reaction system included 0.5 μL primer mix, 2 μL RNA template, 0.5 μL RT enzyme mix, 2 μL 5 × RT buffer, 5 μL ddH₂O. In reference to the corresponding unigene sequence, gene-specific primers were designed using the online tool [GenScript Real-time PCR (TaqMan) Primer Design, https://www.genscript.com/ssl-bin/app/primer]. And the efficiency of the primer pairs were checked by serial dilutions of template cDNA as shown in Additional file 7: Figure S3. The cDNA was diluted to 100 ng μL⁻¹ and then used for qRT-PCR test with each gene-specific primers and SYBR® Green Real time PCR Master Mix (Toyobo, Japan) on the Bio-Rad iQ5 real time system. Reactions were conducted at 96 °C for 1 min, 40 cycles of 95 °C for 15 s, 60 °C for 15 s and 72 °C for 45 s.

The expression patterns of nine genes involved in the anthocyanin pathway (Gene ID: Bra013652, Bra019350, Bra027457, Bra037747, Bra017520, Bra017523, Bra026967, Bra016164, and, Bra032635) were analyzed using qRT-PCR. cDNA was synthesized using ReverseTra Ace qPCR RT Kit (Toyobo, Japan) according to the manufacturer’s recommendation. The reverse transcription reaction system contained 2 μL 5 × RT buffer, 0.5 μL primer mix, 0.5 μL RT enzyme mix, 2 μL RNA template, 5 μL ddH₂O. Three biological replicates were applied for each gene expression analysis. Gene-specific primers were designed according to the reference unigene sequence using the online tool [GenScript Real-time PCR (TaqMan) Primer Design, https://www.genscript.com/ssl-bin/app/primer]. The cDNA diluted to 100 ng/μL was used for qPCR assay with each gene-specific primers and SYBR® Green Real time PCR Master Mix (Toyobo, Japan) on the Bio-Rad iQ5 real time system. Reactions were performed at 96 °C for 1 min, 40 cycles of 95 °C for 15 s, 60 °C for 15 s and 72 °C for 45 s. All primers for RT-qPCR are listed (Additional file 1: Table S1).

Total RNA was extracted from PAECs using TRIzol reagent (Invitrogen Carlsbad, CA, USA) and reverse transcribed into first-strand cDNA with a ReverseTra Ace qPCR RT kit (Toyobo Co., Ltd., Osaka, Japan). The cDNA was subjected to quantitative polymerase chain reaction (qPCR) using a Thunderbird SYBR qPCR Mix (Toyobo) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primers were designed on the basis of GenBank sequences (ACVRL-1, NM_009612.3 and GAPDH, NM_001289726.1). qPCR was run in duplicates, and the delta-delta Ct method was applied for quantification.