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).

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

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.

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.

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

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.

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.

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.