M5 hiper realtime pcr super mix

Total RNA was reverse transcribed into cDNA using a reverse transcriptase kit (Zomanbio, Beijing, China). qPCR was conducted with M5 HiPer Realtime PCR Super mix (Mei5bio, Beijing, China) using qTOWER3 G Real Time PCR Systems (Analytik Jena, Jena, Germany). The primer sequences used in the study are listed below. Mus-Npr1: forward-5′-AGA CGA TGG GCA GGA TAG G-3′, reverse-5′-GGA AGG ATG CTG GGA TGA T-3′; Mus-Itgb4: forward-5′-GTC GCC GTC TGG TAA ACA T-3′, reverse-5′-ACC TGG TCT CCA CGA CTC AC-3′; Mus-Actin: forward-5′-AGA GGG AAA TCG TGC GTG AC-3′, reverse-5′-CAA GAA GGA AGG CTG GAA AA-3′. The Ct values were obtained according to the amplification curve. The relative expression of Npr1 and Itgb4 was calculated by the 2^−ΔΔCt method.

The total RNA from the cells was isolated with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and reverse-transcribed by a commercial kit (Zomanbio, Beijing, China). qPCR with M5 HiPer Realtime PCR Super Mix (Mei5bio, Beijing, China) was carried out using qTOWER3 G Real Time PCR Systems (Analytik Jena, Jena, Germany). The primer sets for the human genes are detailed below:
E2F2, 5′-CGC ATC TAT GAC ATC ACC AA-3′ and 5′-CAA ACA TTC CCC TGC CTA C-3′;
CDKN2B, 5′-GAG GAG AAC AAG GGC ATG-3′ and 5′- CTC CCG AAA CGG TTG ACT-3′;
E2F1, 5′-AGA CCG TAG GTG GGA TCA G-3′ and 5′-TAT GGT GGC AGA GTC AGT GG-3′;
CCNA2, 5′- TGT CAC CGT TCC TCC TTG-3′ and 5′-GGC ATC TTC ACG CTC TAT-3′;
CCNB1, 5′-GGA AGA GCA AGC AGT CAG-3′ and 5′-CTA GCC AGT CAA TTA GGA TG-3′;
CCNB2, 5′-CCC TTG CCA CTA CAC TTC-3′ and 5′-AGA GTC AGC TCC ATC AAA TAC-3′;
CDK1, 5′-GAT GTG CTT ATG CAG GAT T-3′ and 5′-TGT ACT GAC CAG GAG GGA-3′;
CDC25A, 5′-GAG GAT GAT GGC TTC GTG-3′ and 5′-ATC GGT TGT CAA GGT TTG TAG T-3′;
FOXM1, 5′-TGG AGC AGC GAC AGG TTA-3′ and 5′-TGC TGT TGA TGG CGA ATT-3′;
MYBL2, 5′-CGG AGC CCC ATC AAG AAA-3′ and 5′-GCA GTT GTC GGC AAG GAT-3′;
ACTIN, 5′-AGA GGG AAA TCGT GCG TGA C-3′ and 5′-CAA GAA GGA AGG CTG GAA AA-3′.
The Ct values were achieved based on the amplification curve. The relative gene expression was estimated by the 2^−ΔΔCt method.

Total RNA was extracted using a total RNA isolation kit (FORE GENE, Chengdu, China) and reverse transcribed into cDNA. RT-qPCR was performed on this cDNA by using M5 HiPer real-time PCR supermix (Mei5bio, Beijing, China). Three independent experiments were performed in duplicate. The mRNA levels of the target genes of each sample were detected and normalized to the mRNA level of the β-actin/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of the sample, and the value of the control group was set to 1.

After extracting total RNA by E.Z.N.A.^® Plant RNA Kit (Omega Bio-Tek, GA, USA), the cDNA templates for RT-qPCR were made with HiScript II reverse transcriptase kit (Vazyme, Nanjing, China). Then, StepOne Real-Time PCR System (Applied Biosystems, Carlsbad, USA) and the 2× M5 HiPer Realtime PCR Super mix (Mei5bio, Beijing, China) were used for following reactions. AtruActin (ACT) was used as an internal control. The primers used in RT-qPCR are listed in Table S9.

Total RNA was extracted with TRIzol reagent. cDNA template was prepared with HiScript II reverse transcriptase kit (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was used to analyse gene expression levels through StepOne Real-Time PCR System (Applied Biosystems, Carlsbad, USA), and 2 × M5 HiPer Realtime PCR Super mix (Mei5bio, Beijing, China) was used in qRT-PCR reactions. Relative quantification was determined using the 2^−ΔΔCt method [96 (link)]. The primers used are listed in Table S4.

Total RNA were isolated from different maize tissues using RNAiso Plus Kit (Takara, China) following the manufacturer’s protocol, and the cDNA was synthesized using M5 Super plus qPCR RT Kit (Mei5bio, China). Quantitative RT-PCR was performed on a CFX96 Touch System (Bio-Rad, United States) using 2 × M5 HiPer Realtime PCR Super Mix (Mei5bio, China) according to the manufacturer’s protocol. The gene expression levels were calculated by the 2^-ΔΔCT method and were normalized against ZmActin2 gene. The specific primers used for qRT-PCR are listed in Supplementary Table 1.

Total RNA was extracted using an M5 Plant RNeasy Complex Mini Kit (Mei5bio, Beijing) according to the manufacturer’s instructions. cDNA was synthesized using the M5 Super Plus qPCR RT kit with gDNA remover (Mei5bio, Beijing). qPCR was conducted using 2 × M5 HiPer Realtime PCR Super Mix (Mei5bio, Beijing) with an ABI QuantStudio 5 instrument (ABI, Waltham, MA, United States). Expressions of TaPR1-1, TaPR1-4, TaPR1-7, TaPR1-9, TaPR1-16, TaPR1-19, and TaPR1-20 were investigated, and the wheat glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Gene ID: AF251217) gene was used to calibrate the expression levels of queried genes, as previously described (Gao et al., 2015 (link); Supplementary Table 1). Data were analyzed using the 2^–ΔΔCT method (Livak and Schmittgen, 2002 (link)). The statistical significance of differences was calculated using one-way analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) with p < 0.05 in SPSS 26.0 (IBM SPSS Statistics, IBM). For each treatment, three technical repeats and three independent biological replicates were used for analysis.