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Reverse-transcription–polymerase chain reaction (RT-PCR) methods

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.

Quantitative RT-PCR methods

PLoS Med. 2006 December; 3(12): e486.

Quantitative RT-PCRExpression of LOX, NRCAM, BNC1, CCNA1, MAF, ALDH1A3, CTSZ, IRX4, MSX1, KLF11, SERPINB5, TKTL1, GAPDH, r18s, and CDKN2A was analyzed by quantitative real-time RT-PCR. Primers and probes were purchased from Applied Biosystems assay-on-demand, with the exception of p16, which was an assay-by-design (Hs00923893_m1) (http://www.appliedbiosystems.com). All samples were run on the Chromo 4 Real Time Detector (MJ Research [http://www.bio-rad.com]) twice, each time in duplicate. We averaged expression of GAPDH and r18s as internal reference genes to normalize input cDNA. Quantitative real-time reverse-transcriptase-PCR (QPCR) was performed in a reaction volume of 20 μl including 1 μl of cDNA. We used the comparative Ct method to compute relative expression values.

RT-PCR验证芯片结果 results

PLoS Med. 2006 December; 3(12): e486.

Figure 9 MSP for Indicated Genes in Ductal Breast Carcinoma DNA for Samples Obtained from UNC The basal phenotype is based on gene expression profiles demonstrated previously and is characterized by the absence of estrogen receptor and a poor prognosis. Other samples are characterized as luminal. Visible bands corresponding to the appropriate size were counted as positive. 100 bp ladder is at far left. M, methylated product; U, unmethylated product.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) methods

Am J Pathol. 2008 July; 173(1): 30–41.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)For determination of RANTES and MCP-1 mRNA expression, a semiquantitative RT-PCR was used, as described previously.36 Total RNA was prepared from kidney homogenates of various groups of mice. After reverse transcription of the RNA, cDNA was used as a template in PCR reactions using gene-specific primer pairs. After quantifying band intensities by using densitometry, the relative steady-state level of mRNA was calculated after normalizing to β-actin. The sequences of the primer sets were as follows: RANTES, 5′-GTGCCCACGTCAAGGAGTAT-3′ (sense) and 5′-GGGAAGCGTATACAGGGTCA-3′ (antisense); MCP-1, 5′-CCCACTCACCTGCTGCTAC-3′ (sense) and 5′-TTCTTGGGGTCAGCACAGA-3′ (antisense). The sequences of the β-actin primer set were described previously.36

HGF does not block p65 NF-κB nuclear translocation but abolishes its binding to the cognate cis-acting element in RANTES promoter. results

Am J Pathol. 2008 July; 173(1): 30–41.

Figure 7 HGF does not block p65 NF-κB nuclear translocation but abolishes its binding to the cognate cis-acting element in RANTES promoter. A and B: HGF did not block the p65 NF-κB nuclear translocation induced by TNF-α. HKC-8 cells were treated with TNF-α or/and HGF as indicated. A: Representative micrographs showed the staining for p65 NF-κB after various treatments. B: The percentages of cell population with p65 nuclear translocation after various treatments are given. *P < 0.05 versus control. C: ChIP assay demonstrated that HGF signaling abolished the binding of p65 to its cis-acting element in the RANTES promoter. Marker, DNA size markers; IgG, precipitated with control IgG.

HGF does not block p65 NF-κB nuclear translocation but abolishes its binding to the cognate cis-acting element in RANTES promoter. results

Am J Pathol. 2008 July; 173(1): 30–41.

Figure 7 HGF does not block p65 NF-κB nuclear translocation but abolishes its binding to the cognate cis-acting element in RANTES promoter. A and B: HGF did not block the p65 NF-κB nuclear translocation induced by TNF-α. HKC-8 cells were treated with TNF-α or/and HGF as indicated. A: Representative micrographs showed the staining for p65 NF-κB after various treatments. B: The percentages of cell population with p65 nuclear translocation after various treatments are given. *P < 0.05 versus control. C: ChIP assay demonstrated that HGF signaling abolished the binding of p65 to its cis-acting element in the RANTES promoter. Marker, DNA size markers; IgG, precipitated with control IgG.

Reverse transcriptase-Polymerase Chain Reaction methods

Reproductive Biology and Endocrinology 2005, 3:37

Reverse transcriptase-Polymerase Chain Reaction Total RNA was extracted from a pooled sample of five mature male kidneys using Tri Reagent?(Sigma). The cDNA was synthesized from 1 ?/SPAN>g of total RNA using the First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Amplification reactions were assembled using oligonucleotides based upon conserved regions in teleost AR (GeneBank accession numbers: AB012095, AB012096, AB017158, AB023960, AF121257, AB025361 and AF326200). These oligonucleotides were: forward (5'-GGGAAACAGAAATACCTGTGTG-3') and reverse (5'-CTCTGCAATCATCTCTGGAAAG-3'). Amplification was conducted for 40 cycles at 94癈 for 30 s, at 40癈 for 1 min and at 72癈 for 1 min using a PTC-200 Thermal Cycler (MJ Research, Waltham, MA, USA). Amplified products were ligated into pGEM?/SUP>-T (Promega, Madison, WI, USA) and recombinant plasmids were isolated using the Wizard?Plus SV Miniprep System (Promega). Cycle sequencing was performed using the DYEnamic ET Terminator Cycle Sequencing Premix Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The reactions were resolved on an ABI Prism?377 DNA Sequencer (Perkin-Elmer, Milano, Italy) and the data obtained were analyzed using EditView (Version 1.01) (Perkin-Elmer).

RT-PCR methods

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.

Caffeine does not inhibit ATP-induced increases in [Ca2+]i in cultured human pulmonary artery myocytes that lack functional expression of ryanodine re results

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).

Reverse Transcription-PCR methods

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'.

Mechanisms of mammary tumor relapse in MTB/TWNT mice. results

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.

Stable transfection. methods

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.

Reverse Transcription Experiments. methods

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′).

RNA Isolation, RT-PCR, and Real-Time PCR methods

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.

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