7900 real time pcr machine

Total RNA was isolated from the leaves of N. benthamiana plants inoculated with CWMV, reverse transcribed (1 μL total RNA per reaction) using the First Stand cDNA Synthesis Kit (Toyobo, Osaka, Japan) followed by quantitative PCR (qPCR) on a 7900 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA) using the AceQ RT-qPCR SYBR Green Master Mix Kit (Vazyme, Nanjing, China). Each qPCR reaction (20µL) contained 10 µL SYBR Green Master Mix, 2 µL cDNA, 0.5 µL of 10 µM forward and reverse primers, and 7 µL ddH₂O. The qPCR conditions were as: 95 °C for 3 min, 40 cycles of 95 °C for 30 s and 60 °C for 30 s, and 72 °C for 30 s. Three biological replicates with four technical repeats were analyzed for each treatment. The expression of the β-Actin gene was used as the internal reference. The primers used in this assay were listed in the supplementary information (Table S6).

Total RNA was extracted from cells by using Trizol reagent (Invitrogen). cDNA was synthesized by using a reverse transcription kit (Roche). TaqMan qPCR was performed in triplicates using a 7900 Real-time PCR machine (Applied Biosystems, Foster City, CA, USA). β-actin mRNA served as an internal control for gene expression. The average of delta Ct numbers was employed to calculate relative gene expression. The 5′ primers, 3′ primers and fluorescent probes matched from the Universal Probe Library (Cat. No. 04688970001, Roche) were: 5′-tgcacactgtgtttcatcgag, 5′-acgctgtgggactgactttc, Probe #74 for TNFAIP3; 5′-atcaggggccaggttttc, 5′-gggccaagcaccatctaat, Probe #13 for PIM1; 5′-ccagctgacaacaggaggag, 5′-cccatgagctccttgtacagat, Probe #3 for SERPINE1; 5′-ggccttgtgaacagatcagc, 5′-ctccggttcctgcacttg, Probe #69 for FOSL1; 5′-gtggacgggcagaatgtta, 5′-cgtggccagaatctccat, Probe #41 for SDCBP2; 5′-gctcctactgtgataagtccttcc, 5′-tgtcgcctgtgtggattct, Probe #10 for ZNF362; and 5′-tcccacccagaatctttaggta, 5′-gccggggttgagattcat, Probe #10 for EHF.

The TRIzol® LS Reagent (Life Technologies-Ambion USA) was used to achieve total RNA extraction from all groups of blood samples following the producer's protocol. By using High-Capacity cDNA Reverse Transcription Kit (Life Technologies/Ambion/USA), reverse transcription of total RNA was shown in a reaction volume of 20 µl (15 µl total RNA, 2 µl RT buffer, 0.2 µl RT random primers, 0.8 µldNTPs mix, 1 µl RNase inhibitor, and 1 µl reverse transcriptase). As a final point, cDNA was held in reserve at -80C° until used. The gene expression was evaluated using certain primers designed with Primer 3 (http://www.ncbi.nlm.nih.gov/tools/primer-blast) (Table 1). To make the standard curve, serial dilutions of cDNA were used. The standard curves were produced for the target gene and endogenous control gene (ABL). Applied-Biosystems 7900 real-time PCR machine was used for quantitative real-time PCR assays in triplicate. The 20 microliters of reaction volume contained 1 µl of primer mixes, 10 µl of SYBR Green master mix, 4 µl of cDNA template, and 5 µl of RNase-free water. Real-Time PCR protocol was as follows: step 1: 50℃ for 2 minutes, step 2: 95℃ for 10 minutes, step 3, in a two-step cycle: 95℃ for 15 seconds and 65℃ for 1 minute repeated for 6 cycles, and step 4 in a two-step cycle process: 95℃ for 15 seconds and annealing at 61℃ for 1 minute repeated for 40 cycles.