Brightgreen 2x qpcr mastermix

Total RNA was extracted from cell cultures using Trizol (Sigma Aldrich, MO, USA). The mRNA levels were determined by RT-PCR, employing the 5X ALL-IN-ONE RT MASTERMIX (cod G486, ABM, Canada). Real-time PCR assays were conducted using BrightGreen 2X qPCR MasterMix (ABM, Canada) and run on a LineGene 9600 instrument (Bioer Technology, PRC). All reagents were used following the manufacturer's instructions.

Total RNA of breast cancer cells was isolated with TRIzol reagent (Invitrogen) and was reverse-transcribed to cDNA using M-MLV reverse transcriptase (Promega). Quantitative PCR was performed using the BrightGreen 2X qPCR MasterMix (abm) on the CFX96 Real-Time System (Bio-Rad). GAPDH was served as the internal control. The primers used in this study are the follows: S6K1, forward 5′-GCCTCCCTACCTCACACAAG-3′, reverse 5′-CCACCTTTCGAGCCAGAAGT-3′; c-Myc, forward 5′-GGCTCCTGGCAAAAGGTCA-3′, reverse 5′-CTGCGTAGTTGTGCTGATGT-3′; cyclin E1, forward 5′-GCCAGCCTTGGGACAATAATG-3′, reverse 5′-CTTGCACGTTGAGTTTGGGT-3′; GAPDH, forward 5′-GCTGAGAACGGGAAGCTTGT-3′, reverse, 5′-GCCAGGGGTGCTAAGCAGTT-3′.

Gene expression of the candidate genes was observed in specific tissue or under cold temperature. RNA was extracted from specific tissues of wheat at heading stage, which were the first leaf from the ground, stem, peduncle, flag leaf, and spike. In order to observe the gene expression under cold temperature, three-leaf stage wheat seedlings grown at 25 °C were exposed to 4 °C. The RNA extraction was conducted using TRIzol (Thermo Fisher Scientific Co., USA) and cDNA was synthesized using the PrimeScript™ 1st strand cDNA synthesis kit (Takara Bio Inc., Japan) according to the manufacturers’ manuals, respectively. Each gene specific PCR primers was designed using the NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi? LINK_LOC=BlastHome) (Table S5). Real-time PCR was performed using BrightGreen 2X qPCR MasterMix (ABM, Canada) in a CFX-96 real-time PCR machine (Bio-Rad, USA). The Ct values obtained for each gene were normalized depending on the internal gene control, and relative gene expression levels were calculated using the 2^−ΔΔCT method as previously described [71 (link)]. Statistical analysis was performed in R and differences in gene expression were evaluated with Student’s t-test or one-way ANOVA (analysis of variance) followed by a Tukey post hoc test.

RNA was extracted from cell samples with Omnizol (EuroClone, Pero (MI), Italy). The mRNA levels were determined by RT-PCR and by 5X All-In-One RT MasterMix (ABM, Richmond, BC, Canada), and the real-time PCR assays were carried out with BrightGreen 2X qPCR MasterMix (ABM, Vancouver, BC, Canada) and run on a LineGene 9600 PCR detection system (Bioer Technology, Hangzhou, China). All of the reagents were used according to the manufacturer’s instructions. We designed primer pairs for RT-PCR reactions with Primer Express software (Applied Biosystems, Milan, Italy) and used the mRNA sequences as templates from the Nucleotide DataBank (National Center for Biotechnology Information, Bethesda, MD, USA) to design primer pairs. Primer sequences are available upon request. We used the 2-ΔΔCT method as a relative quantification strategy for quantitative real-time PCR data analysis.

Total RNA was extracted from cell cultures using a RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. The quality and quantity of isolated RNA were analysed spectrophotometrically using a Nanodrop (Thermo Fisher). We used sequences of mRNAs from the Nucleotide Data Bank (National Center for Biotechnology Information) to design primer pairs for real‐time RT‐qPCR reactions (Primer Express v. 3.0, Applied Biosystems). The primer sequences are reported in Table 2. cDNA synthesis was performed by 5X ALL‐IN‐ONE RT MASTERMIX (ABM). We used appropriate regions of GAPDH cDNA as controls and ran real‐time PCR assays on a LineGene 9600 machine (Bioer Technology). We carried out reactions according to the manufacturer's instructions using a BrightGreen 2X qPCR MasterMix (ABM). We used the 2^−∆∆CT method as a relative quantification strategy for quantitative real‐time PCR data analysis.