Hieff qpcr sybr green master mix kit

Samples were taken 4 hours after the start of the photoperiod. Total RNA and small RNAs were extracted from the samples of plant leaves and roots (without or with nodules) using TRIzol reagent (Aidlab Biotechnologies Co. Ltd., Beijing, China). Total RNA was treated with genomic DNA Wiper Mix (Yeasen Biotech, Shanghai) to remove genomic DNA. cDNA strands were synthesized from the RNAs using a Hifair II first Strand cDNA Synthesis SuperMix for quantitative polymerase chain reaction (qPCR) kit (Yeasen Biotech, Shanghai). qPCR was performed using a Hieff qPCR SYBR Green Master Mix kit (Yeasen Biotech) with gene-specific primers (table S1). GmELF1b was used as an internal control.
Stem loop–specific RT for miRNA in soybean, L. japonicus and M. sativa was performed as described previously (40 (link)). MiR1520d was used as an internal control of miRNA in soybean (40 (link)). U6s were used as internal controls of miRNA in L. japonicus and M. sativa (47 (link)). Quantitative reverse transcription PCR (qRT-PCR) was conducted using a Hieff qPCR SYBR Green Master Mix kit (Yeasen Biotech) with the gene-specific primers listed in table S1.

Following the instructions, RNeasy kits (Qiagen, German) were used to extract total RNA from cells. After obtaining and quantifying RNA, reverse transcription was performed using the Hifair^® Ⅲ first Strand cDNA Synthesis SuperMix kit (YEASEN, China). After cDNA was obtained, the relative quantification of the target gene in cDNA can be completed with the Hieff^® qPCR SYBR Green Master Mix kit (YEASEN, China). GAPDH was used as an internal reference for quantitative calculation. All procedures were performed on Bio-Rad CFX96 Connect and Roche 480Ⅱ instrument. The primer sequences required for quantitative PCR were presented as follows: GAPDH, forward 5′-ACC​CAG​AAG​ACT​GTG​GAT​GG-3′ and reverse 5′-CAC​ATT​GGG​TAG​GAA​CAC-3′; Cathepsin K, forward 5′-CTT​CCA​ATA​CGT​GCA​GCA​GA-3′ and reverse 5′-TCT​TCA​GGG​CTT​TCT​CGT​TC-3′; CTR, forward 5′-TGC​AGA​CAA​CTC​TTG​GTT​GG-3′ and reverse 5′-TCG​GTT​TCT​TCT​CCT​CTG​GA-3′; TRAP, forward 5′-CTG​GAG​TGC​ACG​ATG​CCA​GCG​ACA-3′ and reverse 5′-TCC​GTG​CTC​GGC​GAT​GGA​CCA​GA-3′; NFATc1, forward 5′-CCG​TTG​CTT​CCA​GAA​AAT​AAC​A-3′ and reverse 5′-TGT​GGG​ATG​TGA​ACT​CGG​AA-30′; V-ATPase d2, forward 5′-AAG​CCT​TTG​TTT​GAC​GCT​GT-3′ and reverse 5′-TTC​GAT​GCC​TCT​GTG​AGA​TG-3′; V-ATPase a3, forward 5′-TGG​CTA​CCG​TTC​CTA​TCC​TG-3′ and reverse 5′-CTT​GTC​CGT​GTC​CTC​ATC​CT-3′;

Total RNA was extracted by TRIzol (Invitrogen) according to the manual. Then the RNA was reverse-transcribed to cDNA using the Hifair^® III 1st Strand cDNA Synthesis SuperMix kit (Yeasen) following the manufacturer’s instructions. The concentration and purity of each sample was tested by DS-11 Spectrophotometer (DeNovix). Equivalent amounts of cDNA were used for real-time PCR in a 20 μL reaction mixture using the Hieff^® qPCR SYBR^® Green Master Mix kit (Yeasen). Primers for qRT-PCR were as shown in Supplementary Table S3. The internal reference gene for lncRNA was GAPDH. QuantStudio5 (Applied Biosystems™) was used to perform the real-time fluorescent quantitative PCR, while ProFlex PCR system (Applied Biosystems™) was used to perform reverse-transcription. Relative levels in each sample were calculated based on their threshold cycle (Ct) values, 2^-ΔΔCt method was used. Each sample had three independent repetitions.