Cfx96 qrt pcr thermocycler

Total RNA from cells or mice tissues was purified with the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. M-MLV Reverse Transcriptase (Takara Bio, Tokyo, Japan) was used for 2 μg RNA reverse transcription, and the cDNAs were used as templates for quantitative real-time PCR (qRT-PCR) amplification using TB Green^® Premix Ex Taq™ II (Takara Bio, Tokyo, Japan) to determine the mRNA expression levels. The qRT-PCR CFX 96 thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for the PCR reaction with 20 µL specific primers. The 2−ΔΔCt method was used to calculate the relative quantity of PCR products. The GAPDH gene was standardized. The primers for qRT-PCR are listed in Table 2.

Total RNA was extracted with the Trizol reagent (Invitrogen, Carlsbad, CA, USA), and 2 μg RNA was reverse transcribed using the MMLV Reverse Transcriptase (Takara Bio, Tokyo, Japan). The obtained cDNAs were used as templates for quantitative real-time PCR (qRT-PCR) or PCR amplification to determine the mRNA expression levels specific primers. The housekeeping GAPDH gene was used for normalization. Quantitative real-time PCR was performed using the following primer sets:
IL-1β forward: 5’-CCAGGGACAGGTATGGAGCA-3’, reverse: 5’-TTCAACACGCAGGACAGGTACAG-3’; IL-2: 5’-CAACTCCTGTCTTGCATTGCACTAA-3’, reverse: 5’-AATGTGAGCATCCTGGTGAGTTTG-3’; IL-6 forward: 5’-AAGCCAGAGCTGTGCAGATGAGTA-3’, reverse: 5’-TGTCCTGCAGCCACTGGTTC-3’; TNF-α forward: 5’-CCTCTCTCTAATCAGCCCTCTG-3’, reverse: 5’- GAGGACCTGGGAGTAGATGAG-3’; MAVS forward: 5’- CGGGAGCAGCAGAAATGA-3’, reverse: 5’- GGCAAGATCCTCGAAGCAG-3’; Nsp5 forward: 5’- TGACAGGCAAACAGCACAAG-3’, reverse: 5’- AGCAGCGTACAACCAAGCTAA-3’;and GAPDH forward: 5’-GCACCGTCAAGGCTGAGAAC-3’, reverse: 5’-ATGGTGGTGAAGACGCCAGT-3’, using TB Green^® Premix Ex Taq™ II (Takara Bio, Tokyo, Japan). PCR reactions were performed using the qRT-PCR CFX 96 Thermocycler (Bio-Rad Laboratories, Inc., CA, USA) in a total volume of 20 µL. The relative amount of PCR products was calculated using the 2^{− ΔΔCt} method.

Total RNA was extracted from the ground leaves according to the manufacturer’s instructions (EASY spin RNA Plant Mini Kit, Aidlab, China). Then, 1μg of total RNA was reverse-transcribed into cDNA with HiScript^® II Q RT SuperMix for qPCR (Vazyme, China) following the instructions. The primer sequences were designed based on the corresponding gene sequence by searching the NCBI database (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 20 December 2020) and Beacon Designer 7.9 (Premier Biosoft International, CA, USA) was used to design the primers which were listed in Supplementary Table S1.
Gene expression analysis was performed using a qRT-PCR CFX96 thermocycler (Bio-Rad, accessed on 24 September 2021). The primers are listed in Supplementary Table S1 (qRT-GmMT-II-F and qRT-GmMT-II-R), and the soybean Actin gene (accession No. V00450) was used as a standard control in RT-PCR reactions. The following thermal cycle conditions were used: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 58 °C for 20 s, and 72 °C for 20 s. All reactions were performed in triplicate. Following the PCR, a melting curve analysis was performed. Ct or threshold cycle was used for relative quantification of the input target number. The relative gene expression levels were calculated according to the 2^–ΔΔCt method [33 (link)].

To evaluate the reliability of differentially expressed genes under Cd stress conditions revealed by RNA sequencing, four genes were selected from the important pathways and validated by quantitative real-time RT-PCR (qPCR). The leaf samples were collected from Cd-treated plants at 0, 1, 4, and 24 h intervals, and freeze-dried in liquid nitrogen. Total RNA was extracted from the ground leaves according to the manufacturer’s instructions (EASYspin RNA Plant Mini Kit, Aidlab, China). Extracted total RNA was reverse-transcribed to cDNA in a total volume of 10 μL using the TransScript II cDNA synthesis SuperMix kit (Transgen Biotech, China). The primer sequences were designed based on the corresponding gene sequence by NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 14 July 2022). Gene expression analysis was performed using a qRT-PCR CFX96 thermocycler (Bio-Rad) with GAPDH serving as an internal control. The primers of target genes and GAPDH were diluted in a ChamQ SYBR qPCR master mix (Vazyme Biotech Co., Ltd., Nanjing, China), and the mix was transferred to a 96-well plate. Reactions were performed and relative expression values were calculated according to the 2^−ΔΔCt [55 (link)].