Abi 7500 fast real time pcr

Total RNA was isolated using TRIzol reagent (Life Technologies, USA) and treated with RNA-free DNase (Roche) to eliminate possible DNA contamination. Reverse transcription was carried out by using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific). The Realtime PCR was done with the SYBR Select Master Mix (Applied Biosystems, USA) according to manufacturer’s instructions. The primer set designed for BRV VP7 gene contains BRV-VP7-F (5′-TAA ATG GAT ATC AAT GGG TT-3′) and BRV-VP7-R (5′-AAC GTC AGT AAT TAC CAG C-3′), and the primer set designed for β-actin gene contains β-actin-F (5′-TCG ATC ATG AAG TGC GA CGT G-3′) and β-actin-R (5′-GTG ATC TCC TTC TGC ATC CTG TC-3′). The PCR reaction was performed with ABI7500 Fast Realtime PCR (Applied Biosystems, USA). Relative levels of viral mRNA were calculated using the 2^-ΔΔCT method of relative quantification with β-actin as the internal control for normalization.

RNA extraction from cell lines was performed using TRIzol RNA Isolation Reagent (Invitrogen) by following the manufacturer’s instructions. The RNA was treated with RQ1 Rnase-free DNase (1 U/μl, Promega, Madison, WI, US, # M6101) for 30 minutes at 37 °C. cDNA was prepared with 1 μg of RNA using oligo-dT and random primers, whereas orientation-specific cDNA was prepared with 3 μg of RNA using Ginir- (sense; G1F) or Giniras- (antisense; G1R) specific primers along with primers for internal control Gapdh at 42 °C for 90 minutes using a Reverse Transcription System kit (Promega, # A3500).
Semiquantitative PCR was performed using Taq DNA polymerase (Merck Bioscience; Darmstadt, Germany). Strand-specific real-time PCR was performed using Mesa green master mix (Eurogenetec, Seraing, Belgium) using ABI 7500 Fast real-time PCR (Applied Biosystems, Foster City, CA, US). Primers used for the experiments are listed in S3 Table.

Six candidate genes for drought tolerance were selected to be validated with quantitative real-time PCR (qRT-PCR). Gene-specific (an exon-exon junction) primers were designed by PrimerQuest Tool (Integrated DNA Technologies, IA, United States), and the actin protein gene was used as an internal housekeeping reference for normalization between samples. RNA was extracted with the method described previously, and three biological replications in two separate wells (technical replication) were applied. The cDNA was synthesized by SensiFAS cDNA Synthesis Kit (Meridian Bioscience, United States), and qRT-PCR was performed on ABI 7500 Fast Real Time PCR (Applied Biosystems, CA, United States) using the SensiFAST SYBR Lo-ROX Kit (Meridian Bioscience, United States), following methods described by Wang et al. (2021) (link). The relative fold changes were calculated using the comparative CT method (2^–ΔΔCT). The average value of the two technical replications was considered for each biological replication.

Primers were designed as follows: human HGF, forward, 5ʹ-CTC ACA CCC GCT GGG AGT AC-3ʹ, reverse, 5ʹ-TCC TTG ACC TTG GAT GCA TTC-3ʹ; mouse HGF, forward, 5ʹ-GGC AAG GTG ACT TTG AAT GA-3ʹ, reverse, 5ʹ-CAC ATG GTC CTG ATC CAA TC-3ʹ; human c-Met, forward, 5ʹ-CTG CCT GCA ATC TAC AAG GT-3ʹ, reverse, 5ʹ-ATG GTC AGC CTT GTC CCT C-3ʹ; mouse c-Met, forward, 5ʹ-CCA GCA GCT TCA GTT ACC GG-3ʹ; human, GAPDH, forward 5ʹ-TGC ACC ACC AAC TGC TTA GC-3ʹ, reverse, 5ʹ-GGC ATG GAC TGT GGT CAT GAG-3ʹ; mouse GAPDH, forward, 5ʹ-GAG TTG TCA TAT TTC TCG T-3ʹ, reverse, 5ʹ-TAT GTC GTG GAG TCT ACT GGT-3ʹ. Cells were grown to 60–70% confluence. RNA was extracted using the Mini Prep kit (Qiagen). CDNA was prepared using the TaqMan Reverse kit (Applied Biosystems). All reactions were performed on the ABI7500 Fast Real-Time PCR.
System (Applied Biosystems). mRNA levels of tested genes were normalized to GAPDH according to the following formula: 2^(ΔΔCT), where CT is the threshold cycle.

The target cell monolayers in 12-well plate were rinsed with PBS and harvested for RNA isolation and qPCR as described previously (35 (link)). Total RNA (1 μg) was reverse transcribed into cDNA using High-Capacity RNA-to-cDNA Reverse Transcription Kit (Applied Biosystems). The expression levels of lncRNA, miRNA, and mRNA were quantified using an ABI 7500 Fast real time PCR (Applied Biosystems, Thermo Fisher Scientific). The relative mRNA expression was calculated using the 2^−ΔΔCt method with β-actin or U6 as an internal reference gene. The primers were listed in Table S1.