Rotor gene real time pcr system

RNA was extracted from cells or tissue using TRIzol, DNase I treated (Zymo Research) and quantitated by spectrophotometry (NanoDrop lite, Thermo Scientific); 0.5 µg RNA was reverse-transcribed using random hexamers and subjected to real-time qRT-PCR. qRT-PCR was carried out using SYBR green with 2 µM of each primer, as previously described [12 (link)], with primers as shown in Table 1. Real-time qRT-PCR was performed using a Rotor-Gene real-time PCR system (Qiagen) as follows: 1 cycle of 95 °C for 5 min, 40 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s; and 1 cycle of 72 °C for 60 s followed by melt curve analysis. Quantitative DENV copy number was calculated as described previously from a standard curve of Mon601 DNA. Relative RT-PCR quantitation was determined by the ∆Ct method [18 (link)] for all other genes, with PCR reactions normalized against the reference housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Total cellular RNA (800 ng for PCR array experiments and 250 ng for individual primer based qRT-PCR experiments) was reverse transcribed into cDNA using RT² PCR First Strand Kit (SABiosciences, Valencia, CA). For human type I IFN response RT² Profiler™ PCR array experiments (SABiosciences), 102 μl of the synthesized cDNA was mixed with 1150 μl RT² SYBR Green/ROX qPCR master mix (SABiosciences) in a final volume of 2300 μl. In qRT-PCR, 1 μl of cDNA and 1 μl of gene-specific PCR primer for human IRF7, NF-κB1, TLR7, or MyD88 (10 μM; SABiosciences) were mixed together with RT² SYBR Green/ROX qPCR master mix (SABiosciences) in a final volume of 25μl. The house keeping gene human B2M (SABiosciences) was used in a separate reaction as endogenous reference in order to normalize differences in the amount of input cDNA in each assay. The thermal profile followed was; intial incubation for 10 min at 95°C (AmpliTaq Gold pre-activation), followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. The PCR run was performed on Rotor Gene real-time PCR system (Qiagen, Santa Clarita, CA). Relative expression of each gene was calculated by the 2^-ΔΔCT method, and differences in gene expression were compared to the mean of the healthy group and expressed as fold-change.

Transgenic rice plants at 5–10 leaf stage were inoculated with SCMV-strain A by rubbing carborundum-dusted leaves with extract of SCMV-infected sugarcane leaves in 0.1 M sodium phosphate buffer (pH 7.0). Infected leaves were harvested at 25 days after inoculation (DPI), and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). The presence of SCMV was analyzed using RT-qPCR using a pair of CP and Hc-Pro specific primers: 5′-TTACAACGAAGATGTTTTCC-3′ (CP-F), 5′-CTGAAATAGTAAATACGAGG-3′ (CP-R) and 5′-CACAGAGCACACACCTACC-3′ (Hc-Pro-F), 5′-CCCAAATTCATCATCCGATAG-3′ (Hc-Pro-R). The rice β-tubulin gene (GenBank Accession number # XM_015794238) was used as the internal reference for the RT-qPCR, using the following PCR primers: TUB-F 5′-GCTGACCACACCTAGCTTTG-3′ and TUB-R 5′-AGGGAACCTTAGGCAGCATG-3′. RT-qPCR was performed in three technical replicates in the Rotor-Gene Real-Time PCR System (Qiagen, Hilden, Germany) using Platinum Taq polymerase (Invitrogen, Thermo Fisher Scientific) and SYBR green.

The miScript miRNA PCR array system, which analyzes the expression of 84 miRNAs associated with human inflammatory and auto-immune response (MIHS-105ZR), was performed in RotorGene Real-Time PCR system (Qiagen, Hilden, Germany). The PCR cycling condition was set as follows: 1 cycle at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for the 30 s. The data containing the Ct (cycle of a threshold) values were analyzed by Qiagen web-based PCR array software using the 2^−∆∆Ct formula to compare miRNA expression between groups^{25 (link)} Ct value higher than 35 indicates no expression of miRNA in the sample and was excluded from the analysis. The global mean normalization method was used to normalize the miRNA Cts^{26 (link)}. The miRNAs showing ≥ twofold changes and p-value < 0.05 were considered as differentially expressed.

Total RNA was extracted from ~100mg of roots from Tamaroi and Nax lines using the RNeasy Plant Mini Kit (Qiagen). First-strand cDNA was synthesized using the QuantiTect Reverse Tanscription Kit (Qiagen), which includes the genomic DNA removal step. Relative transcript levels were assayed by real-time PCR analysis using the Qiagen Rotor-gene Real-Time PCR system. The TdSOS1 gene was amplified using two specific primers TdSOS1; 5′SOS (5′-ATTCCCTCAGGTGCTTCGTG-3′) and 3′SOS (5′-TTTCCTCGAGCAACCCAGTC-3′). The wheat actin gene (Genebank Accession No. AB181991.1) was used as an internal control for gene expression. The actin primers were AF (5′-TACACGAAGCGACATACAA-3′) and AR (5′-AATAGAGCCACCGATCCA-3′). RT-qPCR conditions were as follows: 95 °C for 5min, 94 °C for 30s (50 cycles), 58 °C for 30s, and 72 °C for 1min. Amplified products were detected using QuantiNova SYBR Green PCR Kit (Qiagen). Each data point represents six biological replicates in each sample, presented as the mean ±SE. The experiment was repeated three times, with consistent results.