PLoS Med. 2006 October; 3(10): e420.
Real-Time RT-PCRTotal RNA was extracted from cells using RNeasy kit (Qiagen) and was quantified by UV absorbance spectrophotometry. The reverse transcription reaction was performed by using the Superscript First-Strand Synthesis System (Invitrogen) in a final volume of 20 μl containing 2 μg of total RNA, 100 ng of random hexamers, 1× reverse transcription buffer, 2.5 mM MgCl2, 1 mM dNTP, 10 U of RNaseOUT, 20 U of Superscript reverse transcriptase, and DEPC-treated water. Quantitative real-time RT-PCR analyses of human KEAP1, NRF2, GCLc, GCLm, GSR, PRDX1, GSTA3, GSTA2, NQO1, MRP1, and MRP2 were performed by using Assay-on-Demand primers and probe sets from Applied Biosystems. Assays were performed by using the ABI 7000 Taqman system (Applied Biosystems). β-actin was used for normalization.
Am J Pathol. 2008 July; 173(1): 42–56.
Quantitative Real-Time Polymerase Chain Reaction Analysis. FasL mRNA levels were quantified by real-time PCR analysis. Total cellular RNA was extracted from whole lung or cell lysates using Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA (2 μg) was DNase-treated (Turbo DNA-free kit; Ambion, Austin, TX) and reverse-transcribed using the reverse transcriptase2 first strand kit (SuperArray BioScience) according to the manufacturer’s protocols. The cDNA templates were amplified with mouse β-actin (Superarray catalog no. PPM02945A) and FasL (PPM02926A) primer pairs in independent sets of PCR using reverse transcriptase.2 Real-time SYBR Green PCR master mix (Superarray) on an Eppendorf Mastercycler ep realplex (Westbury, NY) according to the manufacturer’s protocols. Each sample was run in triplicate, and mRNA levels were analyzed relative to the β-actin housekeeping gene. Relative gene expression ratios were calculated according to the SuperArray-recommended ΔΔCt protocol.49
Reproductive Biology and Endocrinology 2008, 6:66
Real time RT-PCR The levels of MMP-2 and -9 gene expression were analyzed with real time RT-PCR using a Taqman probe as described previously [29]. Real time PCR primers were designed using sequence data. Partial bovine MMP-2 and -9 mRNA strands were amplified and identified as follows: Total RNA was isolated using ISOGEN (Nippon Gene, Kyoto, Japan), and one microgram of total RNA was subjected to reverse transcription of cDNA with transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim). The PCR fragments were subcloned into a TA vector (Invitrogen). Both strands were sequenced with a DNA sequence kit using a sequencer (prism 377 DNA Sequencer ABI). We used oligonucleotide primers for cDNA cloning and quantitative real-time RT-PCR analysis are listed in Tables 2 and 3. After confirming their sequence identification, their fragments were used as the standard for measuring their relative expression. All other reagents for mRNA analysis were purchased from Sigma-Aldrich Co. (Saint Louis, MI, USA) or Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).
Reproductive Biology and Endocrinology 2008, 6:41
Quantitative real-time RT-PCR (qRT-PCR) One μg of total cellular RNA from each cell line was reverse-transcribed using random hexamers and MultiScribe Reverse Transcriptase in a two-step RT-PCR reaction (Applied Biosystems, Foster City, CA). Primers (Table 1) were designed using 'Primer Express' (Applied Biosystems) to yield a single amplicon; this was verified by dissociation curves. SYBR Green real-time PCR was performed with an ABI Prism 7000 Sequence Detector or Bio-Rad MyiQ Real-Time PCR Detection System. Thermal cycling conditions included pre-incubation at 50°C for 2 min, 95°C for 10 min followed by 40 PCR cycles at 95°C for 15 sec and 60°C for 1 min. Relative transcript levels were calculated using the relative standard curve method (User Bulletin #2, Applied Biosystems) and results were normalized to 18 S rRNA. Data are reported as mean ± SEM and were analyzed by one-way ANOVA (SigmaStat; Systat Software, Inc., Point Richmond, CA). P < 0.05 was considered to represent a significant difference.
J. Biol. Chem., Vol. 282, Issue 52, 37650-37659
Quantitative Real Time PCR—Quantitative real time PCR was performed using the Mx3000P system (Stratagene) with a SyberGreen MasterMix (Applied Biosystems). Each data point was obtained from at least three independent experiments. Transcripts for glyceraldehyde-3-phosphate dehydrogenase were used as a reference. To ensure specific PCR amplification, every real time PCR run was followed by a dissociation phase analysis (denaturation curve). Specific primer sequences are reported in the supplemental materials. The amplicons corresponding to myogenin, mef2A, Ckm, and Tnnt2 were analyzed by agarose gel electrophoresis, isolated, and sequenced to confirm their identity.
J. Clin. Invest. 118(1): 89-99 (2007)
Quantitative real-time PCR. To assess expression of proangiogenic factors in the tumors, RNA extraction, RT-PCR, and first-strand cDNA synthesis for quantitative real-time PCR analysis (Q-PCR) were carried out as described previously (54, 55). Target gene sequences were from the National Center for Biotechnology Information GenBank databases. Q-PCR was performed using an ABI PRISM 7300 sequence detection system (Applied Biosystems). RNA expression was calculated based on a relative standard curve representing 4-fold dilutions of human cDNA. Q-PCR data were expressed as a relative quantity based on the ratio of the fluorescent change observed with the target gene to the fluorescent change observed with 18S ribosomal subunit. Hepatic endothelial cell and MEF RNA samples were isolated with the Qiagen RNeasy kit following the manufacturer’s directions, and Q-PCR was performed as described above, using the Applied Biosystems TaqMan Assay with prevalidated murine probes and primer sets. A dilution series was carried out for each gene, and the 18S ribosomal subunit was used as an internal control.
