primers for rassf1a  (Pyrosequencing Inc)

Journal: International Journal of Molecular Sciences

Article Title: Novel Biomarkers for Inflammatory Bowel Disease and Colorectal Cancer: An Interplay between Metabolic Dysregulation and Excessive Inflammation

doi: 10.3390/ijms24065967

Figure Lengend Snippet: RASSF1A is epigenetically silenced in inflammatory bowel disease (IBD). ( A ) The methylation percentage of the individual CpGs in the RASSF1A promoter in inflammatory bowel disease patients, n = 80, and 14 control blood samples. The individual CpG methylation percentage indicates what percentage of DNA molecules are methylated at this site in the sample. Circled region corresponds to a hot spot in CRC patients . ( B ) Immunohistochemical staining for RASSF1A in descending colon sections of IBD patients reveals loss of expression in IBD, confirming methylation data in ( A ). Left panel, summary of fold change in IHC staining of tissue sections; right panel, representative sections for each patient category. p value < 0.001 for difference between UC/CD/UC+CD vs. non-IBD (n = 10 for non-IBD and 26 for UC, 32 for CD, and 58 for UC+CD).

Article Snippet: The resulting bisulfite-modified DNA was then used as a template to amplify the region of interest, using a biotinylated primer set for the first exon of Rassf1a, provided by Qiagen (cat # PM00416290).

Techniques: Methylation, Control, CpG Methylation Assay, Immunohistochemical staining, Staining, Expressing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Novel Biomarkers for Inflammatory Bowel Disease and Colorectal Cancer: An Interplay between Metabolic Dysregulation and Excessive Inflammation

doi: 10.3390/ijms24065967

Figure Lengend Snippet: Loss of metabolic markers in IBD and a metabolism-directed therapeutic intervention in an IBD mouse model. Immunohistochemical staining for ( A ) (AMPKα1) pT172 and ( B ) insulin in descending colon sections of IBD patients, reveal loss of activation of (AMPKα1) and tissue expression of insulin. Left panel, summary of fold change in IHC staining of tissue sections; right panel, representative sections for each patient category. ( C ) DSS-induced inflammation injury model of acute inflammation was carried out in the DSS-susceptible model, Rassf1a +/− ( 1a +/− ). Metformin was given in the drinking water at 2 g/L (human equivalent dose [HED] = 500 mg/day) from day 3 to day 9 (during peak of inflammation injury). Mice were monitored for piloerection, bloatednesss, tremors, lack of movement, and rectal bleeding. Left panel, Kaplan–Meier curve with n = 13 (DSS/metformin treated) and n = 28 (DSS treated) animals were used for all conditions and p -value between wild type and Rassf1a −/− . was <0.005. Right panel, immunoblot of descending colon lysates from experiment in the left panel, with the indicated antibodies, as well as representative colon sections stained for active RIPK2 (using the pY 474 RIPK2 antibody) upon metformin treatment. For ( A ), p value < 0.001 for difference between UC/CD/UC+CD vs. non-IBD (n = 30 for non-IBD and 26 for UC, 34 for CD, and 60 for UC+CD). For ( B ), p value < 0.001 for difference between UC/CD/UC+CD vs. non-IBD (n = 14 for non-IBD and 14 for UC, 36 for CD, and 50 for UC+CD).

Article Snippet: The resulting bisulfite-modified DNA was then used as a template to amplify the region of interest, using a biotinylated primer set for the first exon of Rassf1a, provided by Qiagen (cat # PM00416290).

Techniques: Immunohistochemical staining, Staining, Activation Assay, Expressing, Immunohistochemistry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Novel Biomarkers for Inflammatory Bowel Disease and Colorectal Cancer: An Interplay between Metabolic Dysregulation and Excessive Inflammation

doi: 10.3390/ijms24065967

Figure Lengend Snippet: Potential correlations between four identified biomarkers of IBD—RASSF1A, (AMPKα1), YAP, and RIPK2. ( A ) Correlations between biomarkers. For top left, r 2 = 0.07 and p value 0.05, n = 60–74; top middle panel, r 2 = 0.70 and p value < 0.0001, n = 45–50; top right, r 2 = 0.60 and p value < 0.001, n = 55–60; bottom left, r 2 = 0.33 and p value 0.0001, n = 45–60; bottom right, r 2 = 0.10 and p value 0.022, n = 60. ( B ) Correlations between biomarkers and duration of disease. For ( B ), p value < 0.001 for all comparisons explored (n = 30–40 for RIPK and (AMPKα1); n = 15–20 for YAP and RASSF1A analysis). ( C ) Correlations between active RIPK2 pY474 and therapy, before biomarker was established. For ( C ), p value > 0.05 for all treatments, compared to No Tx and n = 12–17 patients per drug group and 40 for the No Tx group (all patients with disease for more than 10 years). ( D ) Correlations between active RIPK2 pY474 and histologic staining of tissues during clinical management. For ( D ), p value > 0.05 for comparing RIPK2 pY474 fold change in patients during remission, when compared to patients with moderate to active disease (n = 10–23 in each category and most patients with disease for more than 10 years).

Article Snippet: The resulting bisulfite-modified DNA was then used as a template to amplify the region of interest, using a biotinylated primer set for the first exon of Rassf1a, provided by Qiagen (cat # PM00416290).

Techniques: Biomarker Discovery, Staining

Journal: International Journal of Molecular Sciences

Article Title: Novel Biomarkers for Inflammatory Bowel Disease and Colorectal Cancer: An Interplay between Metabolic Dysregulation and Excessive Inflammation

doi: 10.3390/ijms24065967

Figure Lengend Snippet: Analysis of biomarkers in matched leukocytes and descending colon sections reveal identical trends, suggesting non-invasive analysis. Leukocyte fraction and descending colon sections were obtained from non-IBD and IBD individuals as indicated. These samples were then immunoblotted for RIPK2 pY474 ( A ), (AMPKα1) pT172 ( B ), RASSF1A ( C ), and YAP pY357 ( D ). Left panels, summary of fold change in IHC staining of tissue sections and densitometrically scanned immunoblotted results; right panel, representative sections for each patient category. For ( A ) (leukocytes), p value is <0.001 for CD or UC or CD+UC vs. non-IBD samples (n = 5–8 for non-IBD, UC or CD, and 12 for UC+CD). For ( A ) (colonic tissue), p value is <0.0001 for CD or UC or CD+UC vs. non-IBD samples (n = 24 for non-IBD, n = 4 for UC or CD, and 8 for UC+CD). For ( B ) (leukocytes), p value is <0.02 for CD or UC or CD+UC vs. non-IBD samples (n = 6 for non-IBD, UC or CD, and 12 for UC+CD). For ( B ) (colonic tissue), p value is <0.001 for CD or UC or CD+UC vs. non-IBD samples (n = 20 for non-IBD, n = 4–8 for UC or CD, and 12 for UC+CD). For ( C ) (leukocytes), p value is <0.004 for CD or UC or CD+UC vs. non-IBD samples (n = 11 for non-IBD, 15 for UC, 7 for CD, and 22 for UC+CD). For ( C ) (colonic tissue), p value is <0.0001 for CD or UC or CD+UC vs. non-IBD samples (n = 8 for non-IBD, 11 for UC, 7 for CD, and 18 for UC+CD). For ( D ) (leukocytes), p value is <0.004 for CD or UC or CD+UC vs. non-IBD samples (n = 4–5 for non-IBD, UC or CD, and 9 for UC+CD). For ( D ) (colonic tissue), p value is <0.0001 for CD or UC or CD+UC vs. non-IBD samples (n = 4 for non-IBD, n = 6–8 for UC or CD, and 13 for UC+CD).

Article Snippet: The resulting bisulfite-modified DNA was then used as a template to amplify the region of interest, using a biotinylated primer set for the first exon of Rassf1a, provided by Qiagen (cat # PM00416290).

Techniques: Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Novel Biomarkers for Inflammatory Bowel Disease and Colorectal Cancer: An Interplay between Metabolic Dysregulation and Excessive Inflammation

doi: 10.3390/ijms24065967

Figure Lengend Snippet: Immunohistochemical staining for RASSF1A, RIPK2 pY474, and YAP pY357 in five patients with UC-CRC. Included in this panel is stage of disease and year diagnosed. Patients B and D have since passed away, of CRC-related disease. ( Left panel ), representative sections for each category are shown; ( Right panel ), summary of fold changes for each biomarker explored. For RIPK2, p value < 0.0001 between UC and UC with no CRC, p value = 0.75 for difference between UC and UC in remission, and p value < 0.0001 between UC and CRC (n = 40–60 sections). For RASSF1A, p value < 0.0001 between UC and UC with no CRC, p value < 0.0001 for difference between UC and UC in remission, and p value < 0.0001 between UC and CRC (n = 10–15 sections). For YAP pY357, p value < 0.0001 between UC and UC with no CRC, p value < 0.0001 for difference between UC and UC in remission, and p value = 0.47 between UC and CRC (n = 4–8 sections).

Article Snippet: The resulting bisulfite-modified DNA was then used as a template to amplify the region of interest, using a biotinylated primer set for the first exon of Rassf1a, provided by Qiagen (cat # PM00416290).

Techniques: Immunohistochemical staining, Staining, Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Novel Biomarkers for Inflammatory Bowel Disease and Colorectal Cancer: An Interplay between Metabolic Dysregulation and Excessive Inflammation

doi: 10.3390/ijms24065967

Figure Lengend Snippet: Models for biomarker utilization to monitor and treat IBD. ( A ) Flowchart of importance of RIPK2/RASSF1A/AMPK. Analysis of RASSF1A, AMPK, and RIPK2 could be used to better understand the molecular status of the patient’s colon and as a therapeutic decision making tool for personalized medicine. ( B ) Proposed blood test for IBD and CRC, based on our case study patient and . Whole blood or leukocyte fraction can be obtained and a rapid high throughput ELISA developed, with monoclonal antibodies to ascertain activation levels. The use of an RIPK2 inhibitor and/or AMPK activator (metformin), are two therapeutic options that could benefit IBD patients, based on our study.

Article Snippet: The resulting bisulfite-modified DNA was then used as a template to amplify the region of interest, using a biotinylated primer set for the first exon of Rassf1a, provided by Qiagen (cat # PM00416290).

Techniques: Biomarker Discovery, High Throughput Screening Assay, Enzyme-linked Immunosorbent Assay, Bioprocessing, Activation Assay

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: The frequencies of promoter methylation of 40 tumor suppressor genes between ovarian cysts and ovarian cancer cell lines

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Methylation

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: The differential percentage of promoter methylation in tumor suppressor genes among ovarian cancer stromal progenitor cells from tissues, ovarian cancer tissues, and ovarian cancer stromal progenitor cells from ascites

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Methylation

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: The differential percentage of promoter methylation in tumor suppressor genes among epithelial-like ovarian cancer stromal progenitor cells from tissues, ovarian cancer tissues, and epithelial-like ovarian cancer stromal progenitor cells from ascites

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Methylation

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: The differential percentage of promoter methylation in tumor suppressor genes among mesenchymal-like ovarian cancer stromal progenitor cells from tissues, ovarian cancer tissues, and mesenchymal-like ovarian cancer stromal progenitor cells from ascites

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Methylation

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: The significantly differential percentage of promoter methylation in tumor suppressor genes between ovarian cyst tissues and ovarian cancer tissues

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Methylation

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: The significantly differential percentage of promoter methylation in tumor suppressor genes between serous type ovarian cancer tissues and non-serous type ovarian cancer tissues

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Methylation

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: The relative mRNA expression folds in CCND2, RASSF1A, RUNX3, CDKN2B, and DLC1 were shown in normal ovarian tissue and epithelial ovarian cancer cell lines.

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: The mRNA expression levels in RASSF1A were significantly lower in advanced-stage EOC (n=24) than in benign ovarian cyst tissues (n=24) (P<0.000) and those in early-stage EOC tissues (n=12) (P<0.004).

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: The expected 5-year overall survival for 110 ovarian cancer patients with methylated promoters of RASSF1A1 was significantly worse than those for patients without methylated RASSF1A (56% vs 80%, P=0.022).

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Methylation

Journal: American Journal of Cancer Research

Article Title: Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

doi:

Figure Lengend Snippet: Univariate and multivariate Cox proportional hazard modeling of survival outcomes in 110 ovarian cancer patients

Article Snippet: RASSF1A gene was amplified by a QIAGEN-designed primer set: RASSF1A (Cat No: QT01016134), GAPDH (Cat No: QT01192646) was used as an internal control.

Techniques: Methylation

Journal: BMC Cancer

Article Title: Inhibition of DNA methyltransferase activity and expression by treatment with the pan-deacetylase inhibitor panobinostat in hepatocellular carcinoma cell lines

doi: 10.1186/1471-2407-12-386

Figure Lengend Snippet: Panobinostat affects activity and expression of DNA methyltransferases in vitro . ( A ) Total DNMT activity was evaluated in HepG2 and Hep3B cells treated with 0.1 μM panobinostat for the indicated points in time. Results are the mean remaining DNMT activity ± relative error of three independent experiments and are expressed relative to values of untreated controls with a set value of 1.0 for each point in time. * P < 0.05 vs. untreated controls. ( B ) and ( C ) Quantitative RT-PCR analysis of expression of DNMTs in HepG2 ( B ) and Hep3B ( C ) cells after treatment with 0.1 μM panobinostat. Results were normalized to the GAPDH level of each sample and represent mean ± relative error of three independent experiments and are expressed relative to mRNA levels of untreated controls at each point in time using the set value 1.0.

Article Snippet: QuantiTect Primers for DNMT1, DNMT3a, DNMT3b, APC, RASSF1A and GAPDH were purchased from Qiagen and subjected to quantitative real-time RT-PCR on a LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) using the LightCycler FastStart DNA Master SYBR Green I Kit (Roche Molecular Biochemicals).

Techniques: Activity Assay, Expressing, In Vitro, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Inhibition of DNA methyltransferase activity and expression by treatment with the pan-deacetylase inhibitor panobinostat in hepatocellular carcinoma cell lines

doi: 10.1186/1471-2407-12-386

Figure Lengend Snippet: Regulation of DNA methylation and expression of target genes after panobinostat treatment. DNA methylation of APC ( A ) and RASSF1A ( B ) was detected by quantitative methylation-specific PCR in HepG2 and Hep3B cells treated with 0.1 μM panobinostat. Expression of total mRNA for APC ( C ) and RASSF1A ( D ) was analyzed using quantitative real-time RT-PCR and normalization to GAPDH content of each sample. Results are mean ± relative error of three independent experiments and are expressed relative to the untreated controls with a set value of 1.0. * P < 0.05 vs. untreated controls.

Article Snippet: QuantiTect Primers for DNMT1, DNMT3a, DNMT3b, APC, RASSF1A and GAPDH were purchased from Qiagen and subjected to quantitative real-time RT-PCR on a LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) using the LightCycler FastStart DNA Master SYBR Green I Kit (Roche Molecular Biochemicals).

Techniques: DNA Methylation Assay, Expressing, Methylation, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Inhibition of DNA methyltransferase activity and expression by treatment with the pan-deacetylase inhibitor panobinostat in hepatocellular carcinoma cell lines

doi: 10.1186/1471-2407-12-386

Figure Lengend Snippet: Effect of panobinostat on DNMT and target gene expression in vivo . HepG2 xenograft specimens were analyzed for expression of ( A ) DNMTs after 1, 7 and 28 days of daily i.p. injections of 10 mg/kg panobinostat. Results were normalized to the GAPDH content of each sample and represent mean ± relative error of 5 to 10 independent samples per group and are expressed relative to expression levels of untreated control animals with the set value of 1.0 for each point in time. ( B ) Methylation status and total expression level of APC and RASSF1A were analyzed at day 7 and day 28 of panobinostat treatment. Results are normalized to levels of untreated controls. Methylation of RASSF1A was not detectable in untreated controls and in treated animals at day 7. * P < 0.05 vs. untreated controls.

Article Snippet: QuantiTect Primers for DNMT1, DNMT3a, DNMT3b, APC, RASSF1A and GAPDH were purchased from Qiagen and subjected to quantitative real-time RT-PCR on a LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) using the LightCycler FastStart DNA Master SYBR Green I Kit (Roche Molecular Biochemicals).

Techniques: Targeted Gene Expression, In Vivo, Expressing, Control, Methylation

Journal: Carcinogenesis

Article Title: Aberrant DNA hypermethylation patterns lead to transcriptional silencing of tumor suppressor genes in UVB-exposed skin and UVB-induced skin tumors of mice

doi: 10.1093/carcin/bgq282

Figure Lengend Snippet: Analyses of the methylated and unmethylated status of DNA and the mRNA expression of the p16INK4a and RASSF1A genes in UVB-exposed epidermal skin and UVB-induced tumors obtained after 24 weeks of the photocarcinogenesis protocol. (A) DNA was extracted and processed for sodium bisulfite conversion and the levels of methylated and unmethylated DNA were determined using real-time PCR. (B) Total RNA was isolated and the mRNA transcripts of the p16INK4a and RASSF1A genes quantified using the real-time PCR SYBR Green system. Data are presented as a relative change in mRNA transcript levels in UVB-exposed epidermal skin and UVB-induced tumors as compared with the levels in the skin of unexposed control mice and are expressed as mean values ± SD; n = 6. Significant difference versus control skin, *P < 0.001.

Article Snippet: Standardized real-time polymerase chain reaction (PCR) primers for Dnmt1, Dnmt3a, Dnmt3b, p 16 INK4a and RASSF1A were obtained from SuperArray Biosciences Corporation (Fredrick, MD).

Techniques: Methylation, Expressing, Real-time Polymerase Chain Reaction, Isolation, SYBR Green Assay, Control

Journal: Carcinogenesis

Article Title: Aberrant DNA hypermethylation patterns lead to transcriptional silencing of tumor suppressor genes in UVB-exposed skin and UVB-induced skin tumors of mice

doi: 10.1093/carcin/bgq282

Figure Lengend Snippet: Modifications in histones and methyl-binding proteins are associated with the DNA hypermethylation of the p16INK4a and RASSF1A promoters in UVB-exposed epidermal skin after 24 weeks. (A) Chip analyses of Ac-histone H3, Ac-histone H4, MeCp2, MBD1 and HDAC1 in the p16INK4a and (B) RASSF1A promoter regions. The panels show the PCR amplification product of the ChIP-purified DNA using the specific antibodies. Samples incubated with mouse IgG served as negative controls. (C and D) Quantification by real-time PCR using the same experimental procedures, antibodies and primers. Data were normalized to input samples and are presented as relative change in DNA and expressed in terms of mean values ± SD (n = 3, epidermal skin or tumor samples were used from three different mice per group). Significant difference versus non-UVB-exposed epidermal skin (normal skin) samples, ***P < 0.05, **P < 0.01, *P < 0.001.

Article Snippet: Standardized real-time polymerase chain reaction (PCR) primers for Dnmt1, Dnmt3a, Dnmt3b, p 16 INK4a and RASSF1A were obtained from SuperArray Biosciences Corporation (Fredrick, MD).

Techniques: Binding Assay, Amplification, Purification, Incubation, Real-time Polymerase Chain Reaction