Stratagene mx3000p real time pcr system

Total RNA was isolated from MAC-T cells treated with HDAC inhibitors using the RNeasy mini kit (Qiagen, Hilden, Germany). The total RNA was then treated with DNase I, then reverse transcription was performed at 42°C for 90 min using 5 μg of total RNA, random primers, and Superscript II RNase H-Reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Comparative real-time PCR was performed using 20 μL of reaction solution containing 10 μL of TOPreal qPCR 2X PreMIX (Enzynomics, Daejeon, Korea), 40 ng of cDNA and each primers (bCSN2-F[10 pmole]; AGCCATGAAGGTCCTCA TCC, bCSN2-R[20 pmole]; CTCACTGCTTGAAAGGC TTT). Each reaction was conducted in triplicate and run in a Stratagene Mx3000P Real-Time PCR system (Agilent Technologies, Inc., Santa Clara, CA, USA). The thermal cycling conditions were as follows; 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 10 min, and extension at 72°C for 1 min. Next, the mRNA levels were corrected using the transcription level of the mouse riposomal protein lateral stalk subunit P0 gene as an internal standard.

Total RNA was isolated from frozen hepatic tissue samples using TRIzol reagent (Thermo Fisher Scientific, Inc., MA, USA) following the manufacturer's protocol. Total RNA was reverse-transcribed, and qPCR was conducted using the TransScript Green Two-Step qRT-PCR SuperMix (TransGen Biotech Co., Ltd., Beijing, China) on a Stratagene Mx3000P Real-Time PCR System (Agilent Technologies, Inc., CA, USA). The relative fold changes in the mRNA levels of the target genes, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), procollagen I (Col-I), and procollagen III (Col-III), were determined using the 2^−ΔΔCt method [26 (link)]. β-Actin was used as a housekeeping gene. All primers are listed in Table 1.

Microarray results were additionally validated using quantitative real time-polymerase chain reaction (qRT-PCR) on RNA samples also used for the microarrays. Custom probes were designed and specificity checked using the National Institutes of Health Primer-BLAST tool and obtained from Integrated DNA Technologies (Coralville, IA). Samples were placed in an Agilent Technologies Stratagene Mx3000P real-time PCR System (Santa Clara, CA) with MxPro software with thermal cycling at 95 °C for 10 min, followed by 40 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. Additionally, RNA samples were analyzed using locked nucleic acid PCR (LNA) miRNA to measure the expression of miRNA targets (A549: miR-25-5p, -378j, -4516; IMR90: miR-149-3p, -608, -4458). Custom LNA probes were obtained from Qiagen (Hilden, Germany). Samples were analyzed with the Mx3000P and MxPro software as per the qRT-PCR experiments except that thermal cycling was performed at 95 °C for 2 min (initial activation), followed by 40 cycles of denaturing at 95 °C for 10 s, and annealing/extension at 56 °C for 1 min. Three independent reactions were performed for each qRT-PCR and LNA PCR experiment with three technical replicates prepared in each. Quantitative data for qRT-PCR and LNA PCR experiments were normalized to the rRNA 5S gene.

Quantitative RT-PCR was performed as described previously [20 (link)]. Briefly, total RNA was isolated using Trizol (Invitrogen, USA). Total RNA (1 μg) was reverse-transcribed into cDNA using the Bestar™ qPCR RT Kit (DBI Bioscience, China). The qRT-PCR reaction was conducted in a total volume of 20 μl containing 10 μl DBI Bestar^® SybrGreen qPCR Master Mix (DBI Bioscience), cDNA derived from 0.2 μg of input RNA, 5 pM of each primer, and 7 μl double-distilled H₂O. PCR reactions were carried out using a Stratagene Mx3000P Real-Time PCR system (Agilent Technologies, USA) with the following steps: pre-denaturation at 95 °C for 2 min, followed by 40 cycles of 94 °C for 20 s, 58 °C for 20 s, and 72 °C for 30 s. Each reaction was performed three times. Fold differences in cDNA level relative to the GAPDH level were calculated using the 2^−ΔΔCt method. The following primers were used: IL-2Rα sense, 5′-AAATGACCCACGGGAAGAC-3′; IL-2Rα antisense, 5′-TTGTGACGAGGCAGGAAGT-3′; LMP1 sense, 5′-CAACAACGGCAAGACTCCC-3′; LMP1 antisense, 5′-CCTCAAAGAAGCCACCCTC-3′).

DNA extracted from biopsy samples was used for quantitative real-time PCR (RT-PCR) on interleukin 23 (IL-23) gene. DNA was amplified with a Stratagene Mx3000P Real-Time PCR System (Agilent Technologies Italia S.p.A., Milan, Italy). Following Taqman gene expression assays were used: Hs00372324_m1 (IL-23A) and Hs999999 m1 (GAPDH, human glyceraldehyde-3-phosphate dehydrogenase). Human GAPDH was used as the housekeeping gene. PCR amplifications were carried out in a 20 μl total volume: 10 μl of 2 × Premix Ex Taq (Takara, Japan), 1 μl of 20 × TaqMan gene expression assay, 0.4 μl of RoX Reference Dye II (Takara, Japan), 4.6 μl of water, and 4 μl of DNA at following PCR conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. PCR reactions were performed in duplicate. The relative abundance of the expression of each gene was calculated by comparing delta cycle thresholds.

EGFR and KRAS mutational testing was conducted as previously described [29 (link)]. Briefly, mutational testing was performed using the HumanEGFR or KRAS Mutation Qualitative Detection Kit (Beijing ACCB Biotech Ltd., China), which applies quantitative real-time PCR (qRT-PCR) platform combining amplification refractory mutation system (ARMS) primers and TaqMan probes. The sensitive mutations were defined as p.G719S/C/A, p.L858R, p.L861Q and insertions in exon 19. The hotspot mutations in KRAS gene were within codon 12 and 13 including p.G12C/V/S/R/D/A and p.G13D. The assay was carried out according to the manufacturer's protocol using the Stratagene Mx3000P real-time PCR system (Agilent technologies Inc, USA). Presence or absence of mutations was assessed from the fluorescence amplification curve.