Transscript 2 green one step qrt pcr supermix

Expression levels of the ncRNAs were evaluated using quantitative RT-PCR
(qRT-PCR) assay. The assay was performed with TransScript II Green One-Step
qRT-PCR Super Mix (TransGen) using a CFX96 Real-Time PCR Detection System
(Bio-Rad). 50°C for 5 min for reverse transcription reaction and denatured at
94°C for 30 s, followed by 40 cycles of 94°C for 10 s, 60°C for 15 s, 72°C for
10 s. The experiments were carried out for three times for each ncRNA. The
relative quantification of ncRNA expression was determined using the
2^ΔΔCt method. The fold change in expression was
obtained by normalizing to an internal control gene TBG-1. All primers used are listed in Table S12.

The total RNA was extracted from leaf and root tissues after HP and LP treatments using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA (~1 μg) was used to synthesize first-strand cDNA using a reverse transcription kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. cDNA was diluted five-fold with nuclease-free water. RT-qPCR was performed using TransScript II Green One-Step qRT-PCR SuperMix (TransGen, Beijing, China), according to the manufacturer’s instructions. The housekeeping genes ZmActin2 and ZmGAPDH were used as internal controls. The primers used are listed in S1 Table.

To validate the RNA-seq data, we selected four DEGs for qPCR experiments. The cDNA samples were synthesized from DNase-treated RNAs employed in the RNA-Seq using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen Biotech, Beijing). PCRs were performed on Roche LightCycler® 480 system using TransScript® II Green One-Step qRT-PCR SuperMix (TransGen Biotech, Beijing). For each sample, reactions were performed in three replicates. The primers are listed in the Supplementary Table 1. The expression of each gene was normalized to the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as reported previously (Hu et al., 2018 (link)). Unpaired two-tailed Student's t-tests were used for statistical analysis using GraphPad Prism® version 9.0.0 (GraphPad Software Inc., USA).

Quantitative RT-PCR was performed using the Rotor-Q qRT-PCR instrument (Qiagen). Total RNA treated with the Turbo DNA-free™ Kit (Ambion) was used. The levels of miRNAs were quantified using the NCode™ VILO™ miRNA qRT-PCR kit (Invitrogen) and normalized with the U6 small nuclear RNA (U6 snRNA). The expression of corresponding miRNA target genes was measured using TransScript II Green One-Step qRT-PCR SuperMix (Transgen) with beta-actin and GAPDH as internal controls. All reactions, including reverse transcriptions and PCRs, were run in triplicates in at least 3 independent experiments. Primers used are listed in Table S15.

Total RNA for RT-qPCR was harvested with the TRIeasy™ Total RNA Extraction Reagent (Yeasen, China). FastKing gDNA Dispelling RT SuperMix (TIANGEN, China) was used for reverse transcription (RT). An miR-29a-specific RT primer was supplied by GenePharma (Shanghai, China). For analysis of microRNA expression, qRT-PCR was performed using TransScript II Green One-Step qRT-PCR SuperMix (TransGen Biotech, China) on the LightCycler® 480. miR-29a expression level was obtained after, which was relative to U6. All PCR primers for mature miR-29a and U6 were ordered from HuaGene. The 2^-ΔΔCt method was applied to calculate relative miRNA expression. All the samples were tested three times [8 (link)].

DNase I-treated total RNA (300 ng) from the early and late intraerythrocytic stages of P. falciparum 3D7 was reverse transcribed. qRT-PCR was performed with 1∶10 diluted cDNAs, a StepOnePlus™ Real-Time PCR System (Applied Biosystems) and TransStart Green qPCR SuperMix (Transgen) under the following cycling conditions: 95°C for 5 min; 40 cycles of 95°C for 15 s, 55°C for 20 s and 72°C for 20 s; and a final extension at 72°C for 5 min. A melting curve was generated as follows: 95°C for 15 s, 55°C for 1 min followed by a temperature gradient with a ramp rate of 0.5°C/s, and a final denaturation at 95°C for 15 s. The obtained products were also evaluated on a 6% denaturing urea PAGE gel. For relative quantification, a house-keeping gene (Pf-β-actin I or U6 snRNA, as appropriate) was used as an internal control. For the intergenic is-ncRNAs, qRT-PCR analysis was also performed on a Rotor-Gene Q apparatus (QIAGEN) using the supplied software and TransScript II Green One-step qRT-PCR SuperMix (Transgen) according to the manufacturer's instructions. The primers used in these experiments are listed in Table S9 in file S1 and the Ct values found for the real time experiment are presented in Table S10 in file S1.