scholarly journals Rapid competitive PCR determination of relative gene expression in limiting tissue samples

1996 ◽  
Vol 42 (2) ◽  
pp. 227-231 ◽  
Author(s):  
Y H Jiang ◽  
L A Davidson ◽  
J R Lupton ◽  
R S Chapkin
Keyword(s):  
Gene Expression ◽  
Mrna Level ◽  
Internal Standard ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Relative Gene Expression ◽  
Competitive Pcr ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Relative Gene

Abstract Reverse transcriptase (RT)-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of procedures, quantification of RT-PCR products remains difficult, particularly when processing a large number of samples. Therefore, we developed a novel alternative PCR technique that we term "rapid competitive PCR" (RC-PCR), designed to study the relative expression of specific genes in a large number of small tissue biopsies. RC-PCR is characterized by measuring relative gene expression at the mRNA level of two or more samples with a nonradioactive assay based on competitive PCR amplification between identical sequences of internal standard and target cDNA. Only a single reaction tube per sample is used in this technique, and it was validated by comparing RC-PCR of protein kinase C zeta and alpha expression in rat colonic mucosa samples with competitive RT-PCR analysis (requiring 6-8 reaction tubes per sample). We conclude that RC-PCR is a simple, rapid, highly sensitive technique that is capable of detecting less than twofold differences in mRNA expression.

scholarly journals Early upregulation of AR and steroidogenesis enzyme expression after 3 months of androgen-deprivation therapy.

2020 ◽  
Author(s):  
Agus Rizal A.H. Hamid ◽  
Harun Kusuma Putra ◽  
Ningrum Paramita Sari ◽  
Putri Diana ◽  
Saras Serani Sesari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Androgen Deprivation Therapy ◽  
Androgen Deprivation ◽  
Relative Gene Expression ◽  
Steroidogenic Enzymes ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Relative Gene

Abstract Background: Androgen deprivation therapy (ADT) is a standard treatment for advanced prostate cancer (PCa). However, PCa recurrence and progression rates during ADT are high. Until now, there has been no evidence regarding when progression begins. This study evaluated the gene expression of intraprostatic androgen receptor (AR) and steroidogenic enzymes in the early stages of ADT. Methods: Prostate tissue samples were taken from PCa patients with urinary retention who received ADT (ADT-PCa; n=10) and were further subgrouped into ADT ≤12 months (n=4) and ADT >12 months (n=6). The ADT-PCa tissues were then compared with BPH (n=12) and primary (no treatment) PCa tissues (n=16). mRNA for gene expression analysis of AR and steroidogenic enzymes was extracted from formalin-fixed paraffin embedded (FFPE) tissues and analyzed by real-time PCR. Protein expression was evaluated by immunohistochemistry with specific antibodies. Results: AR gene expression was higher in the ADT-PCa group than in the BPH or primary PCa group. Both the ADT ≤12 and > 12 months subgroups had significantly higher relative gene expression levels of AR (p<0.01 and 0.03, respectively) than the primary PCa group. In the ADT-PCa group, AR protein expression showed an increasing trend in the ADT ≤12 months subgroup and was significantly elevated in the ADT >12 months subgroup compared with the PCa group (100%; p <0.01). Half (50%) of the patients in the ADT ≤12 months subgroup were found to have upregulation of AR, and one showed upregulation beginning at 3 months of ADT. A trend toward elevated relative gene expression of SRD5A3 was also apparent in the ADT groups. Conclusion: AR and steroidogenic enzymes are upregulated in ADT-PCa patients as early as 3 months, without PSA elevation. Steroidogenic enzymes, particularly SRD5A3, were also upregulated before PSA rose.

scholarly journals Early upregulation of AR and steroidogenesis enzyme expressions after 3 months of androgen-deprivation therapy.

2020 ◽  
Author(s):  
Agus Rizal A.H. Hamid ◽  
Harun Kusuma Putra ◽  
Ningrum Paramita Sari ◽  
Putri Diana ◽  
Saras Serani Sesari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Androgen Deprivation Therapy ◽  
Early Response ◽  
Androgen Deprivation ◽  
Progression Rate ◽  
Relative Gene Expression ◽  
Gene Expressions ◽  
Steroidogenic Enzyme ◽  
Tissue Samples ◽  
Relative Gene

Abstract Background: Androgen-Deprivation Therapy (ADT) is a standard treatment for advanced prostate cancer (PCa). However, there is a high recurrence or progression rate during ADT. Until now, there is no evidence on when the progression starts. This study would like to evaluate the early response of intraprostatic androgen receptor (AR) and steroidogenic enzyme gene expressions in ADT.Methods: Prostate tissue samples were taken from PCa patients with urinary retention, who had ADT (ADT- PCa; n=10), and further grouped into ≤12 months (n=4) and ADT >12 months (n=6). ADT-PCa group were then compared with BPH (n=12) and primary (no treatment) PCa tissues (n=16). AR and steroidogenic enzyme genes were extracted from Formalin Fixed Paraffin embedded (FFPE) tissues and analysed using rtPCR. Protein expressions were evaluated by immunohistochemistry of specific antibodies. Results: AR gene expression was found higher in ADT-PCa group compared to BPH and primary PCa. Both ADT ≤12 and > 12 months subgroups had significantly higher relative gene expression of AR (p 0.01 and 0.03) compared to primary PCa. AR protein expression in ADT-PCa group showed an increase trend in ADT ≤12 months subgroup and a significantly elevated expression AR protein in ADT >12 months subgroup compared with PCa (100%; p <0.01). Half (50%) of ADT ≤12 months patients were found to have upregulation of AR, and one undergone upregulation from only 3 months of ADT. A trend of elevating relative gene expression of SRD5A3 were also found within the groups given ADT. Conclusion: There are upregulation of AR and steroidogenic enzymes in ADT-PCa patients, as early as 3 months without showing PSA elevation. Steroidogenic enzyme, especially SRD5A3 expression was also showing upregulation before PSA rises.

scholarly journals A152 INVESTIGATING THE ROLE OF NOVEL NUDT15 VARIANT IN AZATHIOPRINE-RELATED MYELOTOXICITY

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 155-157
Author(s):  
Q Wang ◽  
A Wilson
Keyword(s):  
Gene Expression ◽  
Gene Expression Level ◽  
Expression Level ◽  
Lower Amount ◽  
Relative Gene Expression ◽  
Rt Pcr ◽  
Its Gene ◽  
The Impact ◽  
Relative Gene ◽  
Relative Gene Expression Level

Abstract Background Azathioprine (AZA), an immunosuppressant, has classically been used to treat patients with inflammatory bowel disease (IBD). AZA inhibits purine synthesis, and its metabolism occurs via a pathway involving thiopurine methyltransferase (TPMT). While standard TPMT genetic screening is conducted for IBD patients initiating AZA treatment to minimize adverse drug effects (ADE), a majority of patients experiencing ADE have wildtype TPMT. Another gene, NUDT15, has been found to be associated with AZA-related myelotoxicity Aims In this study we report two novel variants in NUDT15 and aim to evaluate the impact of NUDT15 variation on its gene expression. We hypothesize that the mutations found within novel NUDT15 variant are detrimental either to the gene’s expression levels or its translation process, resulting in a lower amount of NUDT15 product present and hence translating to AZA-related myelotoxicity observed clinically. Methods IBD patients experiencing AZA-related myelotoxicity were recruited for this study. Patients were then genotyped and the NUDT15 variants were replicated through site-directed mutagenesis. The NUDT15 variants were subsequently transformed into mammalian cell lines then E. coli cells. DNA products were isolated, and transcription levels were assessed through RT-PCR. Results Patient cohort consisted of 27 AZA-exposed IBD patients who developed myelotoxicity despite their TPMT wildtype genotype. Two novel NUDT15 variants were found. The mutation in one of the variants was placed in 3’ UTR, and hence further research was not pursued. Further analysis was conducted for the variant with mutation in coding region. RT-PCR was conducted to assess and compare gene transcription levels between wildtype and variant NUDT15. Wildtype NUDT15 had a relative gene expression level of 0.8x107, whereas variant NUDT15’s relative gene expression level was at 1.1x107. The two groups were not significantly different in terms of gene expression. Conclusions Contrary to our initial hypothesis, it appears that the mutation in the start codon for variant NUDT15 gene does not significantly impact its gene expression as compared to the wildtype gene. We are currently pursuing protein expression analysis studies to assess for translational deficits possibly present in the novel NUDT15 variant. Funding Agencies SRTP - Schulich School of Medicine

scholarly journals Early upregulation of AR and steroidogenesis enzyme expressions after 3 months of androgen-deprivation therapy.

2019 ◽  
Author(s):  
Agus Rizal A.H. Hamid ◽  
Harun Kusuma Putra ◽  
Ningrum Paramita Sari ◽  
Putri Diana ◽  
Saras Serani Sesari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Early Response ◽  
Relative Gene Expression ◽  
Gene Expressions ◽  
Steroidogenic Enzyme ◽  
Tissue Samples ◽  
Elevated Expression ◽  
Ffpe Tissues ◽  
Relative Gene

Abstract Purpose This study would like to evaluate the early response of intraprostatic androgen receptor (AR) and steroidogenic enzyme gene expressions in ADT. Methods Prostate tissue samples were taken from PCa patients with urinary retention, who had ADT (ADT- PCa; n=10), and further grouped into ≤12 months (n=4) and ADT >12 months (n=6). ADT-PCa group were then compared with BPH (n=12) and primary (no treatment) PCa tissues (n=16). AR and steroidogenic enzyme genes were extracted from Formalin Fixed Paraffin embedded (FFPE) tissues and analysed using rtPCR. Protein expressions were evaluated by immunohistochemistry of specific antibodies. Results AR gene expression was found higher in ADT-PCa group compared to BPH and primary PCa. Both ADT ≤12 and > 12 months subgroups had significantly higher relative gene expression of AR (p 0.01 and 0.03) compared to primary PCa. AR protein expression in ADT-PCa group showed an increase trend in ADT ≤12 months subgroup and a significantly elevated expression AR protein in ADT >12 months subgroup compared with PCa (100%; p <0.01). Half (50%) of ADT ≤12 months patients were found to have upregulation of AR, and one undergone upregulation from only 3 months of ADT. A trend of elevating relative gene expression and protein expression of SRD5A1 and SRD5A3 were also found in one patient with 3 months of ADT. Conclusion There are upregulation of AR and steroidogenic enzymes in ADT-PCa patients, as early as 3 months without showing PSA elevation. Steroidogenic enzyme, especially SRD5A, expression was also upregulated before PSA rises.

scholarly journals Maprotiline treatment differentially influences cardiac β-adrenoreceptors expression under normal and stress conditions

2012 ◽  
Vol 48 (4) ◽  
pp. 793-799
Author(s):  
Natasa Spasojevic ◽  
Predrag Jovanovic ◽  
Sladjana Dronjak
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Antidepressant Treatment ◽  
Heart Diseases ◽  
Pcr Analysis ◽  
Mild Stress ◽  
Rt Pcr ◽  
Tissue Samples ◽  
The Right ◽  
Right Atria

Alterations in cardiac function were observed in antidepressants treated patients and published in several clinical reports. These detected changes could be either a consequence of the treatment or of depression itself, which has already been proved to be a risk factor in heart diseases. In order to determine a possible influence of chronic treatment with norepinephrinergic reuptake inhibitor, maprotiline, on the heart, we investigated gene expression of cardiac β-adrenoceptors both in controls and in animals with signs of depression. The rats were divided into two groups, unstressed controls and those exposed to chronic unpredictable mild stress (CUMS). The groups were further divided into two subgroups, one receiving daily intraperitoneal injections of vehicle (sterile water) and another one maprotiline (10 mg/kg) for four weeks. Tissue samples were collected after the last application. Gene expression of cardiac β1- and β2-adrenoceptor was determined using Real-time RT-PCR analysis. Our results show that in control animals expression of both adrenoreceptors was decreased in the right atria after 4 weeks of maprotiline application. Contrary, the same treatment led to a significant increase in expression of cardiac β1-adrenoceptor in the stressed rats, with no change in the characteristics of β2-adrenoceptor. Our findings might reflect the that molecular mechanisms are underlying factors involved in the development of cardiovascular diseases linked with antidepressant treatment.

Relative Gene Expression in Normal and Tumor Tissue by Quantitative RT-PCR

2003 ◽  
pp. 83-98
Author(s):  
Dennis S. Salonga ◽  
Kathleen D. Danenberg ◽  
Jean Grem ◽  
Ji Min Park ◽  
Peter V. Danenberg
Keyword(s):  
Gene Expression ◽  
Tumor Tissue ◽  
Relative Gene Expression ◽  
Rt Pcr ◽  
Relative Gene
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