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

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).

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.

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Reverse Transcription Experiments. Total RNA was extracted from Arabidopsis leaves by using TRIzol reagent (Invitrogen). After conversion to first-strand cDNA, KAT2 and EF1α were amplified by PCR from the same amounts of cDNA by using the following couples of primers: KAT2-3000 (5′-gcgtcttagacgagttagctcgc-3′) and KAT2-3930 (5′-ccgtgaaataggtagacgttctgaacgattggg-3′) or EF1α-350 (5′-ccaccactggtggttttgaggctggtatc-3′) and EF1α-900 (5′-cattgaacccaacgttgtcacctggaag-3′).

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.

J. Clin. Invest. 118(1): 89-99 (2007)

Stable transfection. The GPC1 antisense construct was prepared by RT-PCR amplification of human placenta cDNA, as described previously (11). Stable transfection of GPC1-AS-1751 into PANC-1 cells was performed by using the lipofectamine method (12), and single clones were isolated after 3-4 weeks. After expansion, cells from each individual clone were screened for expression of GPC1 sense and antisense mRNA by northern blot analysis. Parental PANC-1 cells also were transfected with an empty expression vector carrying the neomycin-resistance gene as a control. Positive clones were routinely grown in selection medium.

J. Clin. Invest. 118(1): 51-63 (2007)

Tumor escape in a Wnt1-dependent mouse breast cancer model is enabled by p19Arf/p53 pathway lesions but not p16Ink4a loss J. Clin. Invest. Michael T. Debies, et al. 118:51 doi:10.1172/JCI33320 [Go to this article.] Figure 1Mechanisms of mammary tumor relapse in MTB/TWNT mice. (A) Mammary expression of putative Wnt pathway target genes. Northern hybridization analyses are shown. Mammary gland RNA came from 5-week-old virgin female MTB/TWNT mice that were either Dox naive (Off Dox) or treated with Dox for 96 hours (On Dox). Tumor RNA came from clonally related outgrowths derived from a Dox-dependent MTB/TWNT tumor that was explanted onto the flanks of Dox-treated host mice. Paired flank explants were harvested during ongoing Dox treatment or after timed Dox withdrawal. (B) Tumor gene expression patterns. Northern hybridization analysis was performed on RNA samples from primary and relapsed MTB/TWNT mammary tumors. Three relapsed DITs expressed Wnt1 transgene in an inducer-independent manner (lanes marked T), and 3 expressed aberrant transcripts encoding activated β-catenin variants (β). (C) Molecular genetic analysis of the Ctnnb1 (β-catenin) gene. Segments of transcripts encoding the regulatory domain of β-catenin were amplified from tumor-derived RNA via RT-PCR and subjected to DNA sequencing. The coding region of mouse β-catenin is shown schematically, with arrows indicating the primers used for RT-PCR placed relative to their approximate annealing sites along the open reading frame. The blowup depicts critical aa residues encoded within exon 3; known hot spots for cancer-associated aa substitutions are in b. Asterisks denote the residue affected by the S33Y mutation identified in 2 DITs. The upper chromatograms show detection of only the wild-type β-catenin allele in an antecedent primary tumor but additional detection of the S33Y allele in a descendant recurrent tumor. The lower panels depict RT-PCR–based detection of an aberrantly spliced β-catenin transcript lacking exon 3 within a relapsed tumor (R) and not within the antecedent primary tumor (P). (D) Tumor histology. Photomicrographs of H&E-stained sections derived from representative primary-relapse tumor pairs. The mode of tumor escape identified for each relapse is indicated. Scale bar: 50 μm.

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. Biol. Chem., Vol. 282, Issue 52, 37429-37435

Reverse Transcription-PCR桾otal RNAs were isolated from MCF7 cells, which were uninduced or induced to express Brg1 siRNA for 3 days along with or without treatment of 0.5 ?FONT size=-2>M doxorubicin for 8 h. First-strand cDNA was synthesized by using iScript according to the instructions of the manufacturer (Bio-Rad). A 160-bp cDNA fragment of p21 was amplified with forward primer 5'-CATGTGGACCTGTCACTGTC-3' and the reverse primer 5'-CCTCTTGGAGAAGATCAGCC-3'.A 225-bp cDNA fragment of actin was measured as a loading control, which was amplified with forward primer 5'-CTGAAGTACCCCATCGAGCACGGCA-3' and the reverse primer 5'-GGATAGCACAGCCTGGATAGCAACG-3'.

JGP, Volume 125, Number 4, 427-440

FIGURE 3. Caffeine does not inhibit ATP-induced increases in [Ca2+]i in cultured human pulmonary artery myocytes that lack functional expression of ryanodine receptors. (A) Original recording shows that application of caffeine (10 mM) could not induce an increase in [Ca2+]i in a cultured human PASMC. In the same cell, however, application of ATP (100 礛) evoked a large response in the continued presence of caffeine. (B) RyR1, RyR2, and RyR3 mRNAs are detected in cultured human PASMCs by RT-PCR. Total RNAs were amplified with oligonucleotide primers for human RyR1, RyR2, and RyR3 mRNAs, as described in MATERIALS AND METHODS. The PCR products were separated by gel electrophoresis, stained with ethidium bromide, and visualized by UV illumination. Predicted sizes of RyR1, RyR2, and RyR3 were 224, 265, and 431 bp, respectively. No product was found if reverse transcriptase was omitted (No RT). (C) ATP (10 礛) induced increases in [Ca2+]i in a cell before and after treatment with caffeine (10 mM).

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.

Reproductive Biology and Endocrinology 2008, 6:49

RT-PCR Total RNA was extracted from cultured cells using the RNeasy kit (Qiagen Inc., Mississauga, ON) according to the manufacturer's procedure. Complementary DNA was transcribed from 1.5 μg total RNA using a First Strand cDNA synthesis kit (Amersham, Oakville, Ont., Canada) and used as a template for polymerase chain reaction (PCR). All PCR primers span introns in order to detect specific mRNA sequences. The forward and reverse primers used were as follows: PBX1 (NM_002585): 5'-CCACGTGATGAATCTCCTGCGAGAG-3' and 5'-TCACTGTATCCTCCTGTCTGGCTGA-3', PBX2 (NM_002586): 5'-CTGGTTTGGCAACAAGAGGATTCGC-3' and 5'-TGGAGGTATCAGAGTGAACACTCCC-3' and MEIS1 (BC043503): 5'-AAGGTGATGGCTTGGACAA-3' and 5'-GGCTGCACTATTCTTCTCCG-3'. The expected size of PCR products using these sets of primers are 627 bp for PBX1a, 414 bp for PBX1b, 411 bp for PBX2, and 259 bp for MEIS1. The amplification reaction was carried out in the linear range of the logarithmic phase unless otherwise specified. Each cycle consisted of denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, extension at 72°C for 60 s and a final extension at 72°C for 5 min in a DNA thermal cycler (Mini cyclerTM, PTC-100 TM, MJ-Research, Bio-Rad). PCR products were verified by sequence analysis.

Blood. 2005 September 1; 106(5): 1574–1580.

Reverse-transcription–polymerase chain reaction (RT-PCR)Total RNA was extracted from 8500 cells sorted from each bone marrow or liver population (Absolutely RNA Nanoprep Kit; Stratagene, La Jolla, CA). DNase treatment of all RNA extracts was included according to the manufacturer's instructions. cDNA was prepared by reverse transcription under standard conditions using random hexamer priming (First-Strand cDNA Synthesis Kit; Amersham Biosciences). PCR amplification of c-kit and glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA was performed using FastStart Taq DNA Polymerase (Roche) and the following primer pairs: c-kit forward, 5′-GCACTTGAGTGCTACACTCTTGCACCT-3′; c-kit reverse, 5′-TCTTCAGAACTGTCAACAGTTGGACAACA-3′; GAPDH forward, 5′-TTCCAGTATGACTCCACTCACG-3′; and GAPDH reverse, 5′-GTTCACACCCATCACAAACATG-3′. Conventional PCR conditions using one primer pair per reaction tube were as follows: 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, 40 cycles. Multiplex PCR to amplify both genes in the same reaction tube was performed under identical conditions with both primer pairs added to the same PCR reaction tube.

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

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).