scholarly journals Gel express: a novel frugal method quantifies gene relative expression in conventional RT-PCR

2022 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohamed Hazman
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Express Method ◽  
Relative Expression ◽  
Crossing Point

Abstract Background Real-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression. Nevertheless, compared with conventional PCR, the real-time PCR system is extremely expensive and not affordable for limited or mid-budget research laboratories. Here, a novel, doable and low-cost recipe (referred to as gel express) is developed to quantify gene expression using conventional RT-PCR assay. The novelty of the gel express method is based on replacing crossing point (CP) values with integrated density (IntDen) values of PCR amplicon bands in real-time PCR regular mathematical formulas. Results In this work, gene expression profiles of two different rice stress-marker genes (OsCYP94C2a and OsLOX8) were quantified in response to mechanical wounding at different time points (0, 30, 60, and 150 min). In the gel express method, the free software ImageJ was employed to measure integrated density (IntDen) values of PCR amplicon bands in agarose gel images. IntDen values were then used instead of crossing point (CP) values according to the following modified formula: [EIntDen(ref)/EIntDen(target)]sample ÷ [EIntDen(ref)/EIntDen(target)]control. Gene relative expression profiles (dynamic expression pattern) quantified by gel express method in both genes were highly comparable with real-time RT-PCR. R2 values were 0.9976 and 0.9975 in OsCYP94C2a and OsLOX, respectively. PCR amplification efficiency (E) for all studied genes could be calculated depending on IntDen values through experimentally designed calibration curves. PCR amplification efficiencies with all studied genes obtained by gel express were all in the accepted range. For better-visualized PCR amplicons thus detectable biological effects between treatments, the number of PCR cycles applied in gel express method (IntCyc) was experimentally estimated to be 29 cycles. Conclusions Gel express is a novel, cost-effective and feasible recipe for quantifying gene relative expression in conventional RT-PCR. The expression pattern quantified by gel express is highly comparable and fits the expression data revealed by the used real-time PCR system.

410. Gene expression analysis in endometriotic lesions using laser capture microdissection

2008 ◽  
Vol 20 (9) ◽  
pp. 90
Author(s):  
L. Fu ◽  
J. E. Girling ◽  
P. A. W. Rogers
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Laser Capture Microdissection ◽  
Stromal Cells ◽  
Expression Profiles ◽  
Specific Gene ◽  
Rt Pcr ◽  
Normal Endometrium ◽  
Rna Quality ◽  
Laser Capture

Previous studies examining gene expression profiles in normal endometrium and endometriotic lesions have used RNA extracted from whole tissue samples. Results from these studies can be difficult to interpret as they reflect expression averaged across several different cell types that may be functionally quite different. The aim of this study was to establish laser capture microdissection (LCM) as a technique to examine gene expression in stromal and epithelial cells from normal and ectopic endometrium. We hypothesised that genes associated with inflammation would be elevated in cells from endometriotic lesions. Full thickness uterine samples were collected during abdominal hysterectomy from normal cycling premenopausal women. Endometriotic lesions were collected during abdominal laparoscopy. Samples were either frozen in OCT or stored in RNAlater for 12 h before freezing. Tissues were immunostained with an antibody against CD10 to identify ectopic endometrial stromal cells before LCM. Endometrial epithelial and stromal cells were collected using the PALM MicroLaser System. RNA quality was accessed using Experion. TGFβ1, MMP1, αSMA, SMAD2 and NFκB mRNA was analysed using real-time RT–PCR. Of the endometriotic samples stored in OCT (n = 58), only 14% (n = 8) had visible endometrial glands. Of these, only 37% (n = 3) had RNA of an acceptable quality for further analysis. However, RNA quality and quantity were dramatically improved in 3 of 5 samples collected in RNAlater. In preliminary studies, expression of TGFβ1 and αSMA mRNA was elevated in endometriotic lesions in comparison to the normal endometrium, whereas NFκB expression did not change. We have shown that RNAlater solution is useful to preserve RNA quality for small clinical endometriotic samples and that immuno-guided LCM-generated homogenous cell populations coupled with real-time RT–PCR can provide valuable insights into cell and disease-specific gene expression in endometriotic lesions.

Early T-Cell Precursor-ALL in Adult T-ALL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 911-911 ◽  
Author(s):  
Martin Neumann ◽  
Sandra Heesch ◽  
Stefan Schwartz ◽  
Nicola Gökbuget ◽  
Dieter Hoelzer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Real Time ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Study Group

Abstract Abstract 911 Introduction: Recently, a small subgroup of pediatric acute T-lymphoblastic leukemia (T-ALL) was described, which is closely associated with the gene expression profile of early T-cell precursors (ETPs). This subtype, termed ETP-ALL, showed a highly unfavorable outcome compared to non-ETP(='typical')-ALL. Based on the results of Coustan-Smith et al. (Lancet Oncology, 2009), the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) and St-Jude Children's hospital modified their treatment in children with ETP-ALL to a more intensive regime including stem cell transplantation. ETP-ALL is characterized by a specific immunophenotype (CD1a-, CD8-, CD5weak with expression of stem cell or myeloid markers). Here we explored the existence of ETP-ALL in adults and further studied the molecular characteristics of this specific T-ALL subtype. Patients and methods: We examined the gene expression profiles of 86 adult T-ALL patients obtained from the Microarray Innovations in LEukemia (MILE) multicenter study (HG-U133 Plus 2.0, Affymetrix, Haferlach et al., JCO in press). In addition, bone marrow of 296 patients from the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL) were analyzed by flow cytometry and expression levels of BAALC, IGFBP7, MN1, and WT1 were determined by real-time-PCR. Results: Using the published list of differentially expressed genes in ETPs (Coustan-Smith et al. 2009) we performed unsupervised clustering analyses of the 86 T-ALL samples. A cluster of 17 samples (19.8%) displayed an ETP-associated gene expression profile and were defined as ETP-ALL. Comparing the gene expression profiles of ETP-ALL and typical T-ALL, 2065 probe sets were differentially expressed in ETP-ALL (FDR 0.05). In addition to genes used for classification, we also identified genes known to be involved in the pathogenesis of T-ALL (e.g. PROM1, BCL2, LMO2, LYL1). In particular, stem cell associated genes such as, BAALC (2.52-fold, p=0.003), IGFBP7 (2.76-fold, p=0.002) or MN1 (3.41-fold, p<0.001) were upregulated in ETP-ALL, whereas HOX11 (45-fold, p=0.004), a marker for thymic T-ALL, was downregulated. An independent cohort of 297 patient samples from the GMALL study group was examined by flow cytometry and real-time PCR. 19 (6.4%) samples revealed the ETP-ALL immunophenotype. As expected, all patient samples were found in the group of early T-ALL, representing 23.5% of all early T-ALLs. There was a significant correlation between a lower leukocyte count at first diagnosis and the classification of ETP-ALL (p=0.001). Gene expression measured by real-time-PCR was performed for genes associated with poor outcome in T-ALL: BAALC (2.11-fold, p<0.001) and IGFBP7 (3.59-fold, p=0.003) were significantly upregulated in the group of ETP-ALL. Similarly, the genes MN1 (4.52-fold, p<0.001) and WT1 (2.76-fold, p=0.036), described as poor prognostic markers in cytogenetically normal AML, were also upregulated in ETP-ALL. Conclusion: In adult T-ALL, a subset of patients shares the gene expression profil and immunophenotype of ETP-ALL, which is in line with recent findings in pediatric patients. The gene expression profile of this subset is significantly correlated to stem cell associated markers predictive for inferior outcome in T-ALL. Interestingly, adverse factors in CN-AML are also aberrantly expressed in ETP-ALL suggesting a myeloid origin of ETPs and indicating a closer relationship between ETP-ALL and AML. The prognostic impact and the determination of the most appropiate set of markers needs to be further investigated. These results support the GMALL strategy to regard early T-ALL patients as high risk with assignment to stem cell transplantation. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership.

Gene expression signature predicts chemoresponse of microdissected papillary serous ovarian tumors

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 5064-5064
Author(s):  
L. Ozbun ◽  
T. Bonome ◽  
M. E. Johnson ◽  
M. Radonovich ◽  
C. Pise-Masison ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Gene List ◽  
Response To Therapy ◽  
Advanced Stage ◽  
Protein Ubiquitination ◽  
Ovarian Cancers

5064 Background: The purpose of this study was to identify a predictive gene signature for chemoresponse in patients with advanced stage papillary serous ovarian cancer. Methods: Expression profiling was performed on 50 chemonaive, microdissected advanced stage papillary serous ovarian cancers using Affymetrix Human Genome U133 Plus 2.0 microarrays. Chemoresistance was defined as disease progression while the patients remained on primary chemotherapy. Nine normal human ovarian surface epithelial (HOSE) brushings were also assessed to quantify normal gene expression levels. Validation was performed by quantitative real time PCR using the HOSE isolates and microdissected ovarian tumor samples. Results: A supervised learning algorithm applied to genes differentially expressed between chemosensitive/resistance tumors (p < 0.001) using leave-one-out cross-validation (LOOCV), identified over 2000 genes associated with tumor chemosensitivity. The chemoresponsive gene list was further refined to 576 genes by including only genes used for all LOOCV iterations. An independent gene list was generated comparing expression profiles of chemoresistant tumors to HOSE. The two lists were compared to identify common genes, generating final classifier list of 75 genes that included genes involved in apoptosis, RNA processing, protein ubiquitination, transcription regulation, and other novel genes. We hypothesized genes identified in both data sets would be predictive and biologically relevant. Of these 75 genes, 20 were validated by real-time PCR. Validated genes were ranked by a univariate t-stat value to further resolve the predictor. 4 multivariate predictor algorithms demonstrated the 10 top ranked validated genes maximixed prediction accuracy (compound covariate, 91%; diagonal linear discriminant analysis, 91%; 3-nearest neighbor, 86%; nearest centroid, 95%). The predictive value of these genes will be evaluated on an independent sample set. Conclusions: Gene expression profiling can distinguish between chemosensitive and chemoresistant ovarian cancers. This signature can predict response to therapy and has identified novel biologically and clinically relevant targets. No significant financial relationships to disclose.

103 GENE EXPRESSION PROFILES OF VITRIFIED MOUSE EMBRYOS DETECTED BY MICROARRAYS

2006 ◽  
Vol 18 (2) ◽  
pp. 160
Author(s):  
S. Mamo ◽  
Sz. Bodo ◽  
Z. Polgar ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Differentially Expressed ◽  
Analysis Tool ◽  
Cell Stage ◽  
Base Medium ◽  
Independent Analysis

Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to examine the transcript variations and identify genes most affected by the treatment. For this, 8-cell-stage embryos were collected from female ICR mice mated with ICR males. The embryos were washed with CZB-HEPES base medium and suspended briefly in equilibrium medium consisting of 4% ethylene glycol (EG) in base medium at room temperature. Following equilibration, the embryos were vitrified in a 35% EG, 0.4 M trehalose, 5% polyvinylpyrrolidone (PVP) solution by means of a solid-surface vitrification (SSV) technique as described earlier (Dinnyes 2000 Biol. Reprod. 63, 513-518). Then 40 embryos each from the control and the vitrified/warmed groups were cultured in CZB medium for 3 h. Total RNAs were extracted from cultured embryos in each group using TRIzol (Invitrogen, Bio-Science, Ltd., Budapest, Hungary), following the manufacturer's instructions. Two rounds of amplification were employed to produce labeled RNA, using low input RNA amplification kit (Agilent Technologies, Kromat, Ltd., Budapest, Hungary) procedures with modifications. Three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22K oligonucleotide slides with subsequent analysis of the results. Moreover, as an independent analysis tool, real time PCR was used with eight designed primers. All of the vitrified embryos were recovered after warming with no morphological signs of cryodamage and used for analysis. The two rounds of amplification yielded 15-16 �g of cRNA. The analysis of repeated hybridizations by Rosetta luminator software (Agilent) showed 20 183 genes and expressed sequence tags (ESTs) that passed the selection criteria and were identified as common signatures in all of the slides. Unsupervised analysis of the gene expression data identified a total of 631 differentially expressed (P < 0.01) genes. However, to support the reliability of the results, only those variations above 1.5 fold differences were considered as significant in the final analysis. Therefore, with this stringent criterion 183 genes were differentially expressed (P < 0.01), of which 109 were up-regulated and the remainder down-regulated. Although genes have multiple and overlapping functions, most of the differentially expressed genes were functionally classified into various physiological categories. These include stress response (8), apoptosis related (6), metabolism (51), temperature response (4), and transcription regulation (15). Moreover, the independent analysis with real time PCR and unamplified samples verified the results of microarray. Thus, based on confirmation of the results by an independent analysis and support by the previous studies for some of the genes, it is possible to conclude that the expression patterns reflect the true biological image of embryos after vitrification, with most effects on stress- and cell metabolism-related genes. This work was supported by EU FP6 (MEXT-CT-2003-59582), Wellcome Trust Foundation (Grant No. 070246), and National Office of Research and Technology (NKTH) (#BIO-00017/2002, #BIO-00086/2002).

Identification of Angiogenesis/Metastases Genes Predicting Chemoradiotherapy Response in Patients With Laryngopharyngeal Carcinoma

2007 ◽  
Vol 25 (11) ◽  
pp. 1369-1376 ◽  
Author(s):  
Ian Ganly ◽  
Simon Talbot ◽  
Diane Carlson ◽  
Agnes Viale ◽  
Ellie Maghami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Cdna Microarray ◽  
Expression Profiles ◽  
External Validation ◽  
Cdna Array ◽  
Disease Free Survival ◽  
Independent Set ◽  
Rt Pcr ◽  
Locoregional Failure

Purpose To identify genes related to angiogenesis/metastasis that predict locoregional failure in patients with laryngopharyngeal cancer (LPC) undergoing chemoradiotherapy (CRT) treatment. Methods Tumor tissue was collected and snap-frozen from 35 sequential patients with histologically confirmed LPC being treated with CRT. Gene expression analysis was performed using a novel cDNA array consisting of 277 genes functionally associated with angiogenesis (n = 152) and/or metastasis (n = 125). Locoregional response was correlated to the gene expression profiles to identify genes associated with outcome. These genes were internally validated by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and validated externally by immunohistochemistry analysis on an independent set of patients. Results Locoregional failure occurred in nine of 35 patients. Seventeen genes from the cDNA microarray correlated with locoregional failure (two-sample t test, P < .05). Seven genes were chosen for additional analysis based on the availability of antibodies for immunohistochemistry. Of these seven genes, real-time RT-PCR validated four genes: MDM2, VCAM-1, erbB2, and H-ras (Wilcoxon rank sum test, P = .008, .02, .04, and .04, respectively). External validation by immunohistochemistry confirmed MDM2 and erbB2 as being predictive of locoregional response. Controlling for stage of disease, positivity for MDM2 or erbB2 was an independent negative predictor of locoregional disease-free survival. Conclusion Genomic screening by cDNA microarray and validation internally by real-time RT-PCR and externally by immunohistochemistry have identified two genes (MDM2 and erbB2) as predictors of locoregional failure in LPC patients treated with CRT. The role of these genes in treatment selection and the functional basis for their activity in CRT response merit additional consideration.

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