Gene Transcript Assay by Real-Time Rt-PCR in Epithelial Breast Cancer Cells Selected by Laser Microdissection

2004 ◽  
Vol 19 (2) ◽  
pp. 100-108 ◽  
Author(s):  
V. Becette ◽  
S. Vignaud ◽  
C. Régnier ◽  
M. Labroquère ◽  
E. Fourme ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Real Time ◽  
Target Gene ◽  
Laser Microdissection ◽  
Mrna Level ◽  
Cancer Tissue ◽  
Gene Transcript ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Tissue Samples

The cell type heterogeneity within clinical cancer tissue samples may affect the accuracy of gene expression analysis. In order to validate our laser microdissection (LMD) method using the Leica AS LMD system (LEICA Microsystems), we compared the mRNA levels of three major genes involved in breast cancer (ERα, PR, HER2), measured by means of real-time quantitative RT-PCR, in 5000 microdissected malignant epithelial cells and in corresponding bulk tumor ho-mogenates from 14 patients. We also compared the mRNA level results to protein expression measured by immunohistochemistry (IHC) on the same tumors. For the three genes, significant correlations were found between mRNA results obtained on microdissected cells and IHC. Comparison between IHC and mRNA results obtained on microdissected cells and bulk tumors showed that in all cases microdissection enhanced the sensitivity of assessing target gene transcript levels and was essential for their accurate evaluation in heterogeneous tumors.

scholarly journals Analysis of prolactin receptor expression in breast cancer subtypes

2020 ◽  
Vol 66 (1) ◽  
pp. 89-94
Author(s):  
T.S. Kalinina ◽  
V.V. Kononchuk ◽  
S.V. Sidorov ◽  
L.F. Gulyaeva
Keyword(s):  
Breast Cancer ◽  
Triple Negative ◽  
Prolactin Receptor ◽  
Mrna Level ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Luminal A ◽  
Her2 Positive ◽  
Tissue Samples ◽  
Luminal B

Breast cancer (BC) is the most common cancer among women. It is known that the prolactin receptor (PRLR) may play a role in breast carcinogenesis, but the available data are often contradictory. To get a more complete picture of the relationship between the receptor and mammary gland carcinogenesis, we examined the association between changes in PRLR expression level and tumor subtype (and its main characteristics). To do this, using real-time PCR, we evaluated the level of PRLR mRNA in BC tissue samples and untransformed adjoining tissue samples (89 pairs). Since the androgen receptor (AR) has begun to be seen as a prognostic marker in breast cancer, we also evaluated the association between mRNA levels of AR and PRLR. We found a significant increase in PRLR expression in luminal subtypes; the highest level of PRLR mRNA was detected in luminal A subtype. In HER2-positive ER-, PR-negative BC, the PRLR mRNA level decreases in tumor tissues compared with untransformed tissues. High PRLR expression is also associated with smaller tumor size in luminal B HER2-negative subtype. In ER-, PR-negative tumors, PRLR expression is associated with AR expression: PRLR mRNA level is increased when AR mRNA level is reduced by more than 8 times in triple-negative tumors; in contrast, in HER2-positive subtype it decreases more significantly when AR expression is reduced by more than 3 times. A tendency towards an increase in PRLR expression with an increase in the AR mRNA level was also discovered in luminal subtypes. The level of PRLR expression depends on the age of patients. In luminal A, PRLR expression is higher in patients under 65 years. In contrast, in luminal B HER2-negative and triple-negative BC, reduced PRLR expression was observed in patients under the age of 40 years and under the age of 50 years, respectively. In this group of patients under the age of 40 years with luminal B HER2-negative BC, ER expression was also reduced (0-4 score according to the IHC assay). Thus, PRLR probably plays a different role in the development and progression of BC: in luminal A and luminal B HER2-positive subtypes PRLR may act as an oncogen, and in luminal B HER2-negative and ER-, PR-negative subtypes can play a tumor suppressor role.

Monitoring CML28 mRNA Levels in Patients before and Post HSCT by Real-Time Quantitative RT-PCR.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4478-4478
Author(s):  
Donghua Zhang ◽  
Min Dai ◽  
Hongsheng Zhou ◽  
Yaya Wang ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Mrna Level ◽  
Conditioning Regimen ◽  
Sybr Green ◽  
Sybr Green I ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Standard Samples ◽  
Hematopoietic Stem ◽  
High Level

Abstract A SYBR Green I real-time quantitative RT-PCR method was established for investigating the correlation between CML28 mRNA expressing levels and relapse of leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitorting of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph+ ALL. The sensitivity of the established method was at 10−4 level, with interassay variation and intraassay variation of standard samples both < 10%. The CML28 was highly expressed in AML and CML-BP or AP. In newly diagnosed group, CML28 was (6.58±2.34)×10−2. In pre-conditioning regimen group was (2.19±0.32)×10−2, in group that 1 month after allo-HSCT was (1.35±1.28)×10−2, in group that 3 months after allo-HSCT was (4.57±6.39)×10−3. CML28 can be detected 3months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2×10−2) survived without relapse, the other 2 patients with high level (>2×10−2) relapsed within one year,1 died and1 received the second time allo-HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2×10−2 and relapsed again. CML28 mRNA level was obviously correlated with the development of diseases. Serial quantification of CML28 mRNA levels were necessary for allo-HSCT recipients, and more informative than a single detection. Use of this assay to evaluate MRD in the patients performed allo-HSCT was helpful for predicition of relapse.

scholarly journals Alterations of glutathione and GSTM1 mutations induce tumor metastasis and invasion via EMT pathway in breast cancer patients

2021 ◽  
Author(s):  
Arshad Ali ◽  
Ayaz Ali ◽  
Shafiq Ahmad
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Tumor Metastasis ◽  
Genetic Mutation ◽  
Breast Cancer Tissue ◽  
Cancer Tissue ◽  
Breast Cancer Patients ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Gstm1 Genotype

Abstract Purpose: Alteration in the Glutathione (GSH) and Glutathione S-Transferase (GST) family lead to various disorders including breast cancer. However, the role of GSH and GSTM1 in the onset of breast cancer is still not fully elucidated. Objective: In the present study we observed considerable deficiency in the levels of glutathione and genetic mutation in the GSTM1 enzyme that influence susceptibility to breast cancer metastasis and invasion via EMT pathway. Methods: GSTM1 genotype was identified by multiplex polymerase chain reaction (PCR), RT-PCR and western blotting in breast cancer tissue samples and ANCT samples. The endogenous glutathione levels were determined by HPLC. The tumor metastasis, invasion and EMT biomarkers were determined by RT-PCR and western blot. The relationship between breast cancer, disease progression and histological status were estimated by one way analysis of variance and descriptive statistic. Data were analyzed using OriginPro 2015 statistics software (OriginLab, Northampton, USA). The correlation among different factors was assessed at 95% confidence intervals (CI) using the Mann-Whitney, Kruskal Wallis, and ANOVA test. P<0.05 was considered significant. Results: In present study genotyping analysis of GST investigated that genetic mutation in GSTM1 was detected in breast cancer tissue samples. Moreover, messenger RNA and protein analysis showed that GSTM1 was significant downregulated in tumor tissues (p=0.005, p=0.02) of breast cancer patients. Furthermore, significant reduction in the level of total glutathione level (GSHt P<0.05) was observed among correlation with patient ages, stages and histological grades, of breast cancer patients. Additionally, the result revealed that downregulation of GSTM1 promotes EMT pathway that leads to enhanced the expression of tumor proliferation, invasion and metastasis in breast cancer tissue samples compared with the ANCT samples (P<0.05). Conclusions The present findings suggest that GSTM1 genotype could be a potential biomarker that regulate EMT pathway associated with breast cancer prognosis.

scholarly journals BRCA1 Gene Expression in Breast Cancer: A Correlative Study between Real-time RT-PCR and Immunohistochemistry

2005 ◽  
Vol 53 (5) ◽  
pp. 621-629 ◽  
Author(s):  
Fahd Al-Mulla ◽  
Mahera Abdulrahman ◽  
Govindarajulu Varadharaj ◽  
Nadeem Akhter ◽  
Jehoram T. Anim
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Protein Expression ◽  
Real Time ◽  
Brca1 Gene ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Brca1 Protein ◽  
Brca1 Mrna ◽  
Cancer Tissues

Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard.

scholarly journals Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas

2002 ◽  
pp. 795-802 ◽  
Author(s):  
F Fallo ◽  
V Pezzi ◽  
L Barzon ◽  
P Mulatero ◽  
F Veglio ◽  
...  
Keyword(s):  
Real Time ◽  
Secretory Activity ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Pathophysiological Role ◽  
Aldosterone Production

BACKGROUND: The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. METHODS: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. RESULTS: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. CONCLUSIONS: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

scholarly journals Exercise-induced changes in β-adrenergic-receptor mRNA level measured by competitive RT-PCR

1997 ◽  
Vol 82 (6) ◽  
pp. 1926-1931 ◽  
Author(s):  
Nobuharu Fujii ◽  
Takeshi Shibata ◽  
Sachiko Homma ◽  
Haruo Ikegami ◽  
Kazuo Murakami ◽  
...  
Keyword(s):  
Adrenergic Receptor ◽  
Human Lymphocytes ◽  
Mrna Level ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Receptor Mrna ◽  
Exercise Induced ◽  
Induced Changes ◽  
Receptor Mrna Level

Fujii, Nobuharu, Takeshi Shibata, Sachiko Homma, Haruo Ikegami, Kazuo Murakami, and Hitoshi Miyazaki. Exercise-induced changes in β-adrenergic-receptor mRNA level measured by competitive RT-PCR. J. Appl. Physiol. 82(6): 1926–1931, 1997.—Competitive reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to clarify whether dynamic exercise-induced increases in β-adrenergic-receptor (β-AR) number in human lymphocytes are accompanied by increases in the β-AR mRNA level. Sixteen healthy subjects performed cycle ergometry until exhaustion. Before and immediately after exercise, peripheral blood was drawn from a forearm vein for preparation of lymphocytes. Both the β-AR mRNA level and the β-AR number were significantly increased by exercise. The changes in β-AR mRNA level and β-AR number were significantly correlated ( r = 0.63, P < 0.01). This finding suggests that a rapid increase in β-AR mRNA level might be an early adaptive response of the sympathetic nervous system to dynamic exercise. In vitro incubation of lymphocytes with epinephrine had no effect on β-AR mRNA levels, nor did adenosine 3′,5′-cyclic monophosphate, protein kinase C, or intracellular Ca2+ increase the β-AR mRNA level in vitro. Therefore, it appears that other mechanisms underlie the exercise-induced elevation of β-AR mRNA levels in human lymphocytes.

Strategy for SRM-based Verification of Biomarker Candidates Discovered by iTRAQ Method in Limited Breast Cancer Tissue Samples

2012 ◽  
Vol 11 (8) ◽  
pp. 4201-4210 ◽  
Author(s):  
Satoshi Muraoka ◽  
Hideaki Kume ◽  
Shio Watanabe ◽  
Jun Adachi ◽  
Masayoshi Kuwano ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Tissue ◽  
Cancer Tissue ◽  
Tissue Samples
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