Real-Time PCR Gene Expression Profiling of Microarray Gene Signatures in Acute Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4401-4401
Author(s):  
Ebrahim Sakhinia ◽  
Mahboubeh Farahangpour ◽  
John A. Liu Yin ◽  
Gerard Brady ◽  
Judith A. Hoyland ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Cyclin D3 ◽  
Expression Level ◽  
Tumor Type ◽  
Gene Signatures ◽  
Significant Difference ◽  
The Mean ◽  
Microarray Gene

Abstract Cancer subtype discovery and classification using microarray gene signatures has the potential to transform pathological diagnosis but measurement of indicator genes in routine practice remains difficult. We tested use of real-time PCR measurement of indicator genes for AML and ALL (Golub et al, Science, 1999) as a method for validation and application of microarray gene signatures. Mononuclear cells (MC) were isolated from whole bone marrow (BM) aspirates by density gradient centrifugation and sorted into unselected (total), CD34+ve and CD34-ve fractions. The mRNA in each fraction was globally amplified using a PolyA PCR method. We measured the expression profile of the 17 top ranked genes (cystatin C, leptin receptor, fumarylacetoacetate, CD33, HoxA9, adipsin, proteoglycan 1, LTC4 synthase, LYN, C-myb, MB-1, cyclin D3, SNF2, RbAp48, proteasome iota, HkrT-1 and E2A) from Golub et al (1999) by real-time PCR. All values were calibrated against control standards and normalized to the mean of three housekeeping genes (IF2-beta, GAPDH and human ribosomal protein S9). Data for all 17 genes were obtained for 4 (ALL), 26 (AML), 12 (AML remission) and 9 (morphologically normal) BM samples, each fractionated into three fractions (total MC, CD34+ve MC & CD34−ve MC). There was no significant difference in the mean of three housekeeping gene expression levels between the diagnostic groups. Comparison of the expression level of the other genes confirmed ability to separate AML and ALL, whilst the direction of expression change (increased or decreased) for each gene between AML and ALL was the same as found by Golub et al. In particular, c-myb showed largest significant increase in ALL vs AML in the total BM fraction, whilst cystain c was increased in AML in the CD34−ve fraction. hSNF2b was significantly increased in the ALL total B.M fraction and Hox-A9 was significantly increased in the AML CD34+ve B.M fraction. Furthermore expression level of LYN and CD33 was significantly increased in AML compared to remission AML, indicating ability of the method to determine activity status of disease. In addition, several of the genes provided better separation between AML and ALL when measured in the CD34+ve and −ve fractions indicating more prominent expression in cells of different maturity and that prior fractionation is diagnostically more informative. The results demonstrate ability of the method to validate gene expression signatures by an independent method, which is simple, sensitive and robust, allowing translation to routine clinical use. Whilst the present study used AML and ALL, in principle the method could be extended to any other tumor type for which gene signatures exist.

Clinical Measurement of Prognostic Immune Signature in Follicular Lymphoma by RT-PCR Based Gene Expression Profiling and Immunohistochemistry Demonstrates Favourable T-Cell and Unfavorable Macrophage Infiltration.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 359-359
Author(s):  
Richard J. Byers ◽  
Ebrahim Sakhinia ◽  
Preethi Joseph ◽  
Caroline Glennie ◽  
Sara McDermott ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Image Analysis ◽  
Real Time ◽  
High Power ◽  
Real Time Pcr ◽  
High Power Field ◽  
Gene Signatures ◽  
Expression Levels ◽  
Survival Interval

Abstract Gene expression profiling studies have demonstrated immune response gene signatures predictive of outcome in follicular lymphoma (FL) and there is a need for validation of these signatures and for their translation to clinical use. However, measurement of these genes in routine practice remains difficult and to date there have been very few studies validating the hypothesis. We have previously demonstrated the utility of real-time PCR measurement of gene expression levels in globally amplified polyA cDNA as a clinically practical method for translation of gene signatures to clinical use. In this project we extended the method to analysis of immune response signatures in FL. We used real-time PCR to measure expression levels (normalised to the mean of 4 housekeeping genes) of 36 candidate Indicator genes, selected from microarray studies, in polyA cDNAs prepared using polyA PCR (method detailed in Sakhinia et al 2007) from 58 archived human frozen lymph nodes, together with immunohistochemistry for CD3, CD4, CD7, CD8, CD10, CD20, CD21 and CD68 in parallel formalin fixed paraffin embedded tissue samples to measure immune response in FL. Immunohistochemical positivity was measured by a semi-automated image analysis method using spectral unmixing to identify areas of immunopositivity. Kaplan-Mier survival analysis was performed against the normalised real-time PCR expression levels of each of the genes and against the percentage immunohistochemical postivity for CD3, CD4, CD7, CD8, CD10, CD20, CD21; for CD68 survival analysis was performed for cases with either 15 or less or more than 15 CD68 positive cells per high power field (hpf). High levels of CCR1, a marker of monocyte actication, were associated with a shorter survival interval (p<0.02) (figure 1a), whilst immunohistochemistry demonstrated association of high numbers of CD7 positive T-cells with longer survival interval (figure 1b) (p<0.032) and of high numbers of CD68 positive macrophages with a shorter survival interval (figure 1c) (p<0.02). The results confirm the role of the host immune response in outcome in FL and identify CCR1 as a prognostic indicator and marker of immune switch between macrophage and T-cell dominant response. The methods used are clinically applicable, whilst the clinical utility of polyA DNA and real-time PCR for measurement of gene signatures and the strength of this approach as a “molecular block” are confirmed. Kaplan-Meier Survival Plots for upper (3&4) and lower (1&2) quartiles of a) CCR1 expression and b) number of CD7 +ve cells, and c) cases with less then vs greater than or equal to 15 macrophages per high power field CCR1 measured by real-time PCR and CD7 and macrophage numbers by immunohistochemistry and image analysis Kaplan-Meier Survival Plots for upper (3&4) and lower (1&2) quartiles of a) CCR1 expression and b) number of CD7 +ve cells, and c) cases with less then vs greater than or equal to 15 macrophages per high power field CCR1 measured by real-time PCR and CD7 and macrophage numbers by immunohistochemistry and image analysis

scholarly journals Evaluation of GNMT Gene Expression in Prostate Cancer Tissues using Real-Time PCR

2021 ◽  
Author(s):  
Niloofar Dehghani ◽  
Masoud Salehipour ◽  
Babak Javanmard
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Benign Prostatic Hyperplasia ◽  
Real Time ◽  
Real Time Pcr ◽  
Prostatic Hyperplasia ◽  
Expression Level ◽  
P Value ◽  
Cancer Tissue ◽  
Tissue Samples

Introduction: Prostate cancer is the second most common cancer and the leading cause of cancer-related deaths worldwide. In the present study, the expression level of glycine N-methyl transferase gene (GNMT) was investigated in prostate cancer tissue. The GNMT enzyme is encoded by the GNMT gene. Increased GNMT gene expression increases the conversion of glycine to sarcosine and results in the elevated levels of sarcosine in blood and urine. Methods: The expression level of GNMT gene in tissue samples of patients with prostate cancer was compared with those with benign prostatic hyperplasia using Real-Time PCR technique. Results: The GNMT gene expression level increased significantly in prostate cancer patients compared with those with benign prostatic hyperplasia (p-value <0.001). In addition, the expression level of GNMT gene was stage-dependent and  significant increases were observed in all stages of prostate cancer compared with those with benign prostatic hyperplasia (p-value <0.001). Conclusion: The concentration of sarcosine is controlled by GNMT and it seems that increasing the expression level of GNMT gene increases the level of sarcosine concentration. Thus, it appears that increased levels of GNMT expression occur in the early stages of prostate cancer. Therefore, periodic measurement of GNMT expression levels can detect prostate cancer before it forms a cancer cell and invades other tissues.

Routine expression profiling of microarray gene signatures in acute leukaemia by real-time PCR of human bone marrow*

2005 ◽  
Vol 130 (2) ◽  
pp. 233-248 ◽  
Author(s):  
Ebrahim Sakhinia ◽  
Maboubeh Faranghpour ◽  
John A. Liu Yin ◽  
Gerard Brady ◽  
Judith A. Hoyland ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Profiling ◽  
Acute Leukaemia ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Gene Signatures ◽  
Microarray Gene

260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
A. Baji Gal ◽  
S. Mamo ◽  
S. Bodo ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Standard Error ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Mouse Embryos ◽  
Cell Stage ◽  
The Mean

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P &lt; 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

YY1 Expression Predicts Survival in Follicular and Diffuse Large B-Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2033-2033
Author(s):  
Ebrahim Sakhinia ◽  
Caroline Glennie ◽  
Judith A. Hoyland ◽  
Lia Menasce ◽  
John A. Radford ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cyclin B ◽  
Gene Signatures ◽  
Large B Cell Lymphoma ◽  
Hla Dq ◽  
A Cell

Abstract Recent gene expression profiling has identified gene signatures predictive of outcome, Indicator genes, for diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, measurement of Indicator genes in routine practice remains difficult. We have demonstrated utility of real-time PCR measurement of Indicator genes in globally amplified polyA cDNA as a practical method for their clinical analysis (Sakhinia et al 2005). PolyA PCR enables global mRNA amplification from picogram amounts of RNA and the polyA cDNA pool generated is indefinitely renewable, representing a “molecular block”. Real-time PCR measurement of the expression levels of specific Indicator genes then allows gene signatures to be detected in the polyA cDNA. In this project we applied real-time PCR for 36 Indicator genes, to polyA cDNAs prepared from 122 archived human frozen lymph nodes; specifically 63 cases of FL, 25 of DLBCL and 10 reactive lymph nodes were analysed; the remaining 24 cases were FL with either evidence of focal transformation, of subsequent transformation or (4 cases) paired frozen samples of FL and subsequent DLBCL. PolyA RT-PCR was performed on extracted RNA and resultant cDNA probed for 36 candidate Indicator genes (selected from Husson et al 2002, Shipp et al 2002 and Rosenwald et al 2003), by real-time PCR with quantification against human DNA, and normalisation to the mean of four housekeeping genes. Statistical analysis was performed using the Mann Whitney test and Kruskal-wallis with a p≤ 0.05 for statistical significance; all results detailed below are significant to at least p≤ 0.05. Ten genes showed statistically significant different expression between FL and DLBCL, including Cyclin B, COL3A1, NPM3, H731, PKC.B1, OVGL, ZFPC150, HLA-DQ-a, and XPB. Of these, cyclin b, a cell cycle gene, NPM3, nucleolar phosphoprotein, and COL3A1were higher in DLBCL. Six genes showed statistically significant higher expression in the neoplastic nodes compared to reactive nodes, namely PKCB-1, BCL-6, EAR2, ZFX, Cyclin B, YY.1. Comparison of gene expression levels in cases of FL that either did not or did subsequently transform to DLBCL demonstrated significant upregulation of ACTA, HLA-DQ-a in the latter cases of FL. High levels of YY.1 were associated with a shorter survival interval in both FL and DLBCL by Kaplan-meir survival analysis. This is of particular interest given the reported role of YY.1 in producing resistance to Rituximab due to downregulation of FAS -induced apoptosis (Vega et al, 2005). This was reported for a cell culture study and has not been shown in clinical studies, though the result in this project suggests a possible detrimental role for YY.1 clinically and that it may act as a predictor of Rituximab response. These results :demonstrate the possibility of using polyA PCR for global amplification of clinical samples and real-time PCR to measure diagnostically informative gene expression profiles in the resultant polyA cDNA “molecular block” andidentify novel prognostic markers for FL and DLBCL. The method is simple, sensitive and robust, and amy be used as a platform for clinical measuremen of prognostic gene signatures. Whilst lymphoma represents a relatively small group of cancer patients the generic nature of microarray gene profiles for cancer subtypes will facilitate simple extension of the method to other tumour types.

scholarly journals Expression level of DREB gene of local corn cultivars from Kisar Island-Maluku, Indonesia, using quantitative real time polymerase chain reaction

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Hermalina Sinay ◽  
Estri Laras Arumingtyas
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Flag Leaf ◽  
Block Design ◽  
Rna Extraction ◽  
Expression Level ◽  
Total Rna ◽  
Deep Yellow ◽  
Dreb Gene

The research objective was to determine the expression level of dehydration responsive element binding (DREB) gene of local corn cultivars from Kisar Island Maluku, Indonesia. The study was designed as randomized block design with single factor consist of six local corn cultivars obtained from farmers in Kisar Island and one reference varieties which has been released as a drought-tolerant varieties and obtained from Cereal Crops Research Institute Maros South Sulawesi. Isolation of total RNA from the second leaf after the flag leaf at the 65 days after planting were carried out according to the protocols of the R and ABlueTM Total RNA Extraction Kit, and was used as a template for cDNA synthesis. Amplification of cDNA from total RNA was carried out according to the protocol of One-Step Reverse Transcriptase PCR Premix Kit. Real Time-PCR was performed usingcDNA from reverse transcription following the procedures of Real MODTM Green Real-Time PCR Master Mix Kit. The real time-PCR data were analyzed using relative quantification method based on the critical point/cycle threshold. The highest DREB gene expression was showed by Deep Yellow local corn cultivar, and the lowest one was showed by Rubby Brown Cob cultivar. The DREB gene expression level of deep yellow local corn cultivar was even higher than Srikandi variety as a reference variety.

scholarly journals Usefulness of a real-time PCR platform for G+C content and DNA–DNA hybridization estimations in vibrios

2011 ◽  
Vol 61 (10) ◽  
pp. 2379-2383 ◽  
Author(s):  
Ana Paula B. Moreira ◽  
Nei Pereira ◽  
Fabiano L. Thompson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Hybridization ◽  
Sister Species ◽  
Wide Range ◽  
Significant Difference ◽  
The Mean ◽  
Vibrio Campbellii ◽  
The Relationship ◽  
Linear Regression Curve

The aim of this study was to evaluate the utility of a real-time PCR platform to estimate the DNA G+C content (mol%) and DNA–DNA hybridization (DDH) values in the genus Vibrio. In total, nine vibrio strains were used to determine the relationship between genomic DNA G+C content and T m (°C). The T m and HPLC datasets fit a linear regression curve with a significant correlation coefficient, corroborating that this methodology has a high correlation with the standard methodology based on HPLC (R2 = 0.94). Analysis of 31 pairs of vibrios provided a wide range of ΔT m values, varying between 0.72 and 12.5 °C. Pairs corresponding to strains of the same species or strains from sister species showed the lowest ΔT m values. For instance, the ΔT m of the sister species Vibrio harveyi LMG 4044T and Vibrio campbellii LMG 11216T was 5.2 °C, whereas the ΔT m of Vibrio coralliilyticus LMG 20984T and Vibrio neptunius LMG 20536T was 8.75 °C. The mean ΔT m values corresponding to pairs of strains with DDH values lower than 60 % or higher than 80 % were, respectively, 8.29 and 2.21 °C (significant difference, P<0.01). The high correlation between DDH values obtained in previous studies and the ΔT m values (R2 = 0.7344) indicates that the fluorimetric methodology is a reliable alternative for the estimation of both DNA G+C content and ΔT m in vibrios. We suggest that strains of the same Vibrio species will have less than 4 °C ΔT m. The use of a real-time PCR platform represents a valuable alternative for the development of the taxonomy of vibrios.

scholarly journals Lead and cadmium influences on the metallothionein expression level in Lymnaea stagnalis L. adults

1970 ◽  
pp. 281-285
Author(s):  
S. E. Dromashko ◽  
S. N. Shevtsova ◽  
A. S. Babenko
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Lymnaea Stagnalis ◽  
Test System ◽  
Expression Level ◽  
Pond Snail ◽  
Cadmium Ions ◽  
Lead And Cadmium ◽  
Expression Evaluation

Aim. The article deals with the assessment of some heavy metals effects on metallothionein expression in such a test system as the great pond snail L. stagnalis. Methods. There are methods of mollusk specimen cultivation and gene expression level estimation (PCR in real-time mode), as well as data analysis. Results. A significant increase in MT gene expression was observed in both young (4.5 months) and older (7 months) mollusks on the 7th day of exposure to 0.003 mg/L of lead ions as well as significant increase in MT gene expression was observed in older mollusks on the 7th day of exposure to 0.0001–0.001 mg/L of cadmium ions. Conclusions. The expediency of L. stagnalis adults using at the age of 3 to 5 months with other test systems for biotesting liquid metal-containing waste based on the gene expression evaluation in a short (no more than 7 day) experiment using the Real Time PCR has been shown.Keywords: Lymnaea stagnalis, lead/cadmium, metallothioneins, gene expression, real time PCR.

Sign in / Sign up

Export Citation Format

Share Document