A method of quantitative assessment of cytokine gene mRNA expression levels using real-time PCR in homogenous low cell number populations

2014 ◽  
Vol 50 (4) ◽  
pp. 430-434
Author(s):  
A. M. Savilova ◽  
M. M. Chulkina ◽  
A. S. Speranskaya ◽  
D. Yu. Trofimov
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Assessment ◽  
Cell Number ◽  
Cytokine Gene ◽  
Expression Levels ◽  
Mrna Expression Levels ◽  
Gene Mrna

scholarly journals Simultaneous Quantitative Detection of Relevant Biomarkers in Breast Cancer by Quantitative Real-Time PCR

2006 ◽  
Vol 21 (1) ◽  
pp. 30-39 ◽  
Author(s):  
M. Labuhn ◽  
V. Vuaroqueaux ◽  
F. Fina ◽  
A. Schaller ◽  
I. Nanni-Metellus ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Mrna Levels ◽  
Quantitative Real Time Pcr ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Mrna Expression Levels

The assessment of ERα, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERα, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERα, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Analysis of gene expression in the rat hippocampus using real time PCR reveals high inter-individual variation in mRNA expression levels

2002 ◽  
Vol 67 (2) ◽  
pp. 225-234 ◽  
Author(s):  
Julieta Alfonso ◽  
Guido D. Pollevick ◽  
Anja Castensson ◽  
Elena Jazin ◽  
Alberto C.C. Frasch
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Real Time ◽  
Individual Variation ◽  
Real Time Pcr ◽  
Rat Hippocampus ◽  
Expression Levels ◽  
Mrna Expression Levels

Evaluation of thymidylate synthase and ERCC1 mRNA levels as predictive markers in colorectal cancer patients treated with S-1 and oxaliplatin

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e15071-e15071
Author(s):  
H. Kuramochi ◽  
K. Hayashi ◽  
G. Nakajima ◽  
H. Kamikozuru ◽  
M. Yamamoto
Keyword(s):  
Colorectal Cancer ◽  
Mrna Expression ◽  
Real Time ◽  
Cancer Patients ◽  
Thymidylate Synthase ◽  
Excision Repair ◽  
Mrna Levels ◽  
Expression Levels ◽  
Colorectal Cancer Patients ◽  
Mrna Expression Levels

e15071 Background: Oxaliplatin has been widely used for the treatment of colorectal cancer. The mechanism of action of platinum compounds such as oxaliplatin is to bind to a DNA molecule in the form of a platinum-DNA-adduct. Excision repair cross complementation group 1 (ERCC1), which plays a major role in the nucleotide excision pathway, has a polymorphism in codon 118, and is reported to be associated with a resistance to platinum-based therapy. Thymidylate synthase (TS) and dehydropyrimidine dehydrogenase (DPD) are key enzymes of 5-FU metabolism and are well known to be associated with a response to 5-FU-based therapy. Methods: Twenty-one colorectal cancer patients (male:female = 7:14; median age, 65) treated with a combination of oxaliplatin and S-1 as a first-line therapy were analyzed for ERCC1 codon 118 polymorphism and the mRNA expression levels of TS, ERCC1, and DPD. Formalin-fixed paraffin- embedded surgical specimens were used and t-RNA and DNA were extracted. The mRNA expression levels were measured using real-time RT-PCR, and the polymorphism was analyzed using the allelic discrimination method together with real-time PCR. Results: No correlation was observed between ERCC1 codon118 polymorphism and any response to the chemotherapy. ERCC1 mRNA levels tended to be higher in the patients with wild-type homozygous alleles in codon 118 than in those with at least one mutant allele(1.19 vs.0.68: p= 0.15). Patients with both high TS and ERCC1 mRNA levels showed a significantly lower response rate than the others (25% vs. 67%, p=0.02). No relationship was seen between DPD mRNA expression levels and the response. Conclusions: The mRNA expression levels of TS and ERCC1 appear to be useful markers for the treatment of S-1 and oxaliplatin. No particular usefulness of ERCC1 codon 118 polymorphism was verified. No significant financial relationships to disclose.

scholarly journals Comparative Evaluation on the Expression of Pi3 Kinase - AKT- mTOR Signalling Molecules in Oral Squamous Cell Carcinoma - A Real Time PCR Based Approach

2021 ◽  
pp. 182-188
Author(s):  
Krishnapriya Umashankar ◽  
J. Selvaraj ◽  
Pratibha Ramani
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Squamous Cell ◽  
Genetic Predisposition ◽  
Real Time Pcr ◽  
Cell Cancer ◽  
Genetic Research ◽  
Rna Isolation ◽  
Pi3 Kinase ◽  
Expression Levels

Background: Oral squamous cell cancer (OSCC) develops as a result of the accumulation of many genetic mutations that are influenced by genetic predisposition. Upon acquisition of genetic predisposition, the precancerous cells will transform into malignant cells culminating into carcinomas. Advances in genetic research over the past few decades have rendered early detection possible. Aim: To compare the gene expression of Pi3 Kinase, AKT and mTOR in OSCC and to correlate the expression levels of these molecules with the survival in OSCC patients. Also to understand the role of Pi3 Kinase pathway in OSCC progression thereby attempting targeted therapy in OSCC patients. Materials and Methods: 10 OSCC samples as well as normal healthy samples were collected and RNA isolation was done using RNA easy kit from Qiagen (Valencia, CA), and then subjected to cDNA synthesis using Human TGF-β1, Human GSK-3β and Human Pi3 kinase primers. Real time PCR was performed using gene specific primers at 40 cycles. The results were retrieved, tabulated and analyzed. Results: The current research results revealed that there were up regulation of mRNA expression in The PI3K/AKT/mTOR in OSCC patients than in healthy individuals. On comparison, mTOR showed highest mRNA expression levels than AKT and PI3K. Conclusion: Overexpression of Pi3 kinase, AKT, mTOR plays a crucial role in progression of oral cancer and targeting Pi3 kinase/mTOR pathways could be a novel and targeted approach for OSCC.

Quantitative real time-PCR of CD133 mRNA: A potential surrogate angiogenic marker of response for patients with metastatic sarcoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20034-20034
Author(s):  
E. H. Lin ◽  
Y. Li ◽  
J. Trent ◽  
S. M. Patel ◽  
M. A. Burgess ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Progressive Disease ◽  
Tumor Response ◽  
Mrna Levels ◽  
Paired Samples ◽  
Before And After ◽  
Metastatic Sarcoma ◽  
Mrna Expression Levels

20034 Background: CD133 antigen is a specific surface marker for circulating endothelial progenitor cells (CEPs), pivotal in post-natal angiogenesis. We hypothesize that changes in levels of the CD133 mRNA expression and vascular endothelial growth factor (VEGF) may correlate with tumor response. Methods: After informed consent, we obtained 48 peripheral blood samples from patients with metastatic sarcoma including gastrointestinal stroma tumors (GIST). There were 16-paired samples before and after treatment including chemotherapy, or surgery, or imatinib. Of 24 patients with metastatic GIST enrolled, seven were paired samples. We measured CD133 mRNA levels by method (Lin E et al AACR 2003# 5392) and VEGF levels by ELISA (Genzyme, MA). The measurements were done in duplicates in two experiments. Results: The mean CD133 levels and VEGF levels before and after treatment was in Table 1 . The treatment resulted in significant reduction of CD133 mRNA expression (p = 0.035) as well as the level of VEGF (p = 0.014). Three patients experienced increase in CD133 expression had progressive disease. Among the paired GIST patients, there was a trend of increased CD133 mRNA expression levels in patients with progressive disease, death, or tumor recurrence. CD133 mRNA levels appeared elevated among the imatinib naïve patients and imatinib appeared to reset CD133 mRNA levels among the responding GIST patients to that of healthy volunteers. Conclusion: CD133 mRNA expression levels in sarcoma patients measured by real time-PCR assay appeared to correlate with tumor response. Unpublished independent work with NASBA assay platform in other solid tumors supported our findings ( www.primagen.com ). Further work is needed to reduce assay variability, and to determine the sensitivity and specificity of assay comparing to flow cytometry and to test assay’s applicability in the antiangiogenic therapy of cancer and the angiogenic therapy of cardiovascular diseases. [Table: see text] No significant financial relationships to disclose.

scholarly journals Expression of ITG β-1, MMP9 and Vimentin in Oral Squamous Cell Carcinoma – A Real Time PCR Based Approach

2021 ◽  
pp. 81-87
Author(s):  
Krishnapriya Umashankar ◽  
J. Selvaraj ◽  
Pratibha Ramani
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Cancer ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Mrna Expression ◽  
Real Time ◽  
Squamous Cell ◽  
Real Time Pcr ◽  
Tissue Samples ◽  
Expression Levels

Background: Oral Squamous Cell Carcinoma (OSCC) is the most common malignancies which accounts for 90% of oral cancer worldwide. The 5 year survival rate of OSCC is not more than 60% due to tumor metastasis and subsequent recurrence. Aim: To compare the gene expression of ITG β-1, MMP9 and Vimentin in OSCC tissue samples and normal tissue samples and to correlate the expression levels of these molecules with the pathological grading and survival in OSCC patients. This would facilitate the understanding of EMT in OSCC progression thereby targeting this pathway for treatment of OSCC patients in near future. Materials and Methods: 10 OSCC samples as well as normal healthy samples were collected and RNA isolation was done using TRIR kit, and then subjected to cDNA synthesis using ITG β-1, MMP9 and Vimentin primers. Real time PCR was performed using gene specific primers at 40 cycles. The results were retrieved, tabulated and analyzed. Results: The current research results revealed that there were up regulation of mRNA expression in ITG β-1, MMP9 and Vimentin in OSCC patients than in healthy individuals. On comparison, MMP9 showed highest mRNA expression levels than ITG β-1 and Vimentin Conclusion: Over expression of ITG B-1, MMP9, Vimentin plays a crucial role in progression of oral cancer and targeting EMT molecules could be an effective targeted approach for OSCC.

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