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, 6:66

Gelatinase mRNA expression profiles obtained with quantitative PCR during gestation The microarray data suggest that MMP-2 has an initiating role in endometrial remodeling throughout gestation. Therefore, we focused on gelatinase expression and analyzed the profiles in more detail using quantitative RT-PCR (Fig. 2). The highest MMP-2 expression was found during the estrous cycle, although rather high expression remained in early gestation. During the middle of gestation, MMP-2 expression was decreased and fell to low levels with as gestation progressed. The level was 3- to 4-times less than that observed during the estrous cycle. The expression levels in CAR were higher than those in ICAR during the estrous cycle and early gestation. There were no significant differences between the levels in the intercotyledon and cotyledon. In cotyledonay tissue, MMP-2 expression was lower than in the endometrium throughout gestation.

Reproductive Biology and Endocrinology 2008, 6:66

Gelatinase mRNA expression during gestation as analyzed by quantitative PCR. The expression values were normalized with cloned MMP-2 and MMP-9 as standards. Expression refers to the mean +/- SD. Two cows were used on each day during the estrous cycle, and three cows were used on each day during gestation. The abbreviations are the same as in Figure 1. A: MMP-2 in endometrium. B: MMP-9 in endometrium. C: MMP-2 in fetal side tissues. D: MMP-9 in fetal side tissues. a-c:abc; a-d: a, b, c, d; a-e: a, b, c, d, e; a-f: a, b, c, d, e, f; b-f: b, c, d, e, f; c-f: c, d, e, f; d-f: d, e, f. Different letters above each bar indicate significant difference at P < 0.05. No significant difference was found in Figures 2B, 2C and 2D. Kizaki et al. Reproductive Biology and Endocrinology 2008 6:66 doi:10.1186/1477-7827-6-66

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.

Plant Physiol. 2006 January; 140(1): 302–310

RNA Isolation, RT-PCR, and Real-Time PCRTotal RNA was prepared from plants by using the RNeasy plant minikit (Qiagen). Two micrograms of RNA was used as a template for first-strand DNA synthesis using the SuperScript II first-strand synthesis system for RT (Invitrogen). PCR amplification was performed using Taq DNA polymerase (Invitrogen). Real-time quantitative PCR was run on an ABI 7500 real-time PCR system (Applied Biosystems) according to the manufacturer's recommendations. Real-time quantitative PCR reaction contained 25 μL 2× SYBR Premix Ex Taq (TaKaRa), 2 μL primer mix, 1 μL 50× ROX Reference Dye II, 4 μL cDNA, and 18 μL deionized water to make a total volume of 50 μL. After setting the amplification conditions, experiments were repeated twice. The primers used were as follows: ABA2 (At1g52340), 5′-ctcgctttggctcatttgc-3′ and 5′-ccgtcagttccaccccttt-3′; NCED3 (At3g14440), 5′-ccggtggtttacgacaagaa-3′, and 5′-cccaagcgttccagagatg-3′; and Actin2 (At3g18780), 5′-gctgagagattcagactgccca-3′ and 5′-cacagttttcgcgatccagac-3′. For relative quantification the method of Pfaffl (2001) was used to determine the relative expression ratio. This determines the expression of the target gene relative to reference gene (ACTIN2) in a test sample compared with an untreated Col sample.

J Clin Invest. 2007 July 2; 117(7): 2004–2013.

RNA isolation and real-time PCR. Total RNA was extracted from cerebellar lysates using the TriFast method (peqGold TriFast; PEQLAB) following the manufacturer’s instructions. First-strand cDNA was synthesized in a volume of 20 μl using 1 μg of total RNA and TaqMan reverse transcription reagents (Applied Biosystems). The RNA was quantified at 260 nm in a NanoDrop spectrophotometer at an OD 260/280 ratio of 1.7 to 2.0 for all samples. PCR was performed with an ABI PRISM 7000 (Applied Biosystems) and 2 × qPCR MasterMix (Eurogentec) according to the manufacturers’ instructions. To quantify target mRNA levels, TGF-β, TNF-α, IFN-γ, IL-10, and IL-4 TaqMan Gene Expression assays were purchased from Applied Biosystems. The sequences CXCL12 (forward) 5′-CCAGAGCCAACGTCAAGCA-3′, CXCL12 (reverse) 5′-TGCACACTTGTCTGTTGTTGTTCTT-3′, and CXCL12 (probe) 5′-Fam-CTCAACACTCCAAACTGTGCCCTTCAGA-TAMRA-3′ were designed with Primer Express software (version 2.0; Applied Biosystems). All primers spanned an intron to ensure discrimination between cDNA and genomic DNA. The relative amount of specific mRNA was normalized to GAPDH using the following sequences: GAPDH (forward) 5′-CTCAACTACATGGTCTACATGTTCCA-3′; GAPDH (reverse) 5′-CCATTCTCGGCCTTGACTGT-3′; and GAPDH (probe) 5′-Fam-TGACTCCACTCACGGCAAATTCAACGT-TAMRA-3′. All PCR reactions were run in duplicate and were performed with 40 cycles, including a negative control consisting of PCR-grade water. Quantitative real-time PCR analysis was carried out using the 2-ΔΔCt method (55).