Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells

AbstractBackgroundQuantitative real time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) are methods used for gene expression analysis in several contexts, including reproductive endocrinology.ObjectivesHerein, we compared qPCR and ddPCR technologies for gene expression analysis of hormone membrane receptor-encoding genes, such as follicle-stimulating hormone (FSHR), G protein-coupled estrogen (GPER) and choriogonadotropin receptors (LHCGR), as well as the commonly used RPS7 housekeeping gene, in order to identify the most reliable method to be applied for gene expression analysis in the context of human reproduction.MethodsTotal RNA was extracted from human primary granulosa cells of donor patients undergoing assisted reproduction and used for gene expression analysis by qPCR and ddPCR, after finding the optimal annealing temperature.ResultsBoth techniques provided results reflecting the low number of FSHR and GPER transcripts, although ddPCR detected also unspecific transcripts in using RPS7 primers and quantified the low-expressed genes with major accuracy, thanks to its higher reaction efficiency. The absolute FSHR and GPER transcript number was also determined by ddPCR, resulting in 50- to 170-fold lower amount than LHCGR transcripts.ConclusionThese results suggest that ddPCR is the candidate technology for analysis of genes with relatively low expression levels and provides useful insights for characterizing hormone receptor expression levels in the context of reproductive endocrinology.

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Effect of TRIM28 on proliferation, apoptosis and histone H3K9 trimethylation in bovine fibroblasts

Indian Journal of Animal Research ◽

10.18805/ijar.b-883 ◽

2019 ◽

Author(s):

Panpan Ma ◽

Xiaxia Man ◽

Shubao Yang ◽

Xiaoqing Dong ◽

Liyan Su ◽

...

Keyword(s):

Gene Expression ◽

Polymerase Chain Reaction ◽

Cell Proliferation ◽

Real Time ◽

Chain Reaction ◽

Expression Levels ◽

H3k9 Trimethylation ◽

Polymerase Chain ◽

Apoptosis Gene ◽

Hp1 Proteins

TRIM28 is a co-repressor, which interacts with HP1 proteins and chromatin repressive complexes leading to gene expression silencing. In this study, after we used Lipofectamine3000-mediating siRNA and restructured p EGFP-IRES2-TRIM28 to transfect bovine fibroblasts, the expression levels of TRIM28, HP1BP3, DNMT, SETDB1, TP53, BAX, Bcl and SOD were detected by real-time polymerase chain reaction (PCR). In addition, the influence of p EGFP-IRES2-TRIM28 on histone H3K9me3 was also observed. In the siRNA transfected group, TRIM28 was down-regulated (P less than 0.01), which inhibited cell proliferation and reduced the level of H3K9me3 expression. In the p EGFP-IRES2-TRIM28 transfected group, TRIM28 was over-expressed (P less than 0.01), and the pro-apoptosis gene BAX was significantly decreased (P ess tahn 0.01), but there was no significant change in other genes. Our results demonstrate that TRIM28 plays an important role in bovine fibroblasts and might be a valuable biomolecule for proliferation, apoptosis, and histone H3K9 trimethylation.

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Genetic transformation of sweet oranges with the D4E1 gene driven by the AtPP2 promoter

Pesquisa Agropecuária Brasileira ◽

10.1590/s0100-204x2013000700006 ◽

2013 ◽

Vol 48 (7) ◽

pp. 741-747 ◽

Cited By ~ 4

Author(s):

Lísia Borges Attílio ◽

Francisco de Assis Alves Mourão Filho ◽

Ricardo Harakava ◽

Tatiane Loureiro da Silva ◽

Luzia Yuriko Miyata ◽

...

Keyword(s):

Gene Expression ◽

Polymerase Chain Reaction ◽

Genetic Transformation ◽

Transgene Expression ◽

Sweet Orange ◽

Chain Reaction ◽

Real Time Quantitative Pcr ◽

Phloem Protein ◽

Polymerase Chain ◽

Gene Expression Levels

The objective of this work was to produce transgenic 'Pêra' and 'Valência' sweet orange plants using the D4E1 gene driven by the Arabidopsis thaliana phloem protein (AtPP2) promoter and to quantify transgene expression in different transformation events. Genetic transformation experiments were carried out with epicotyl segments co‑cultivated with Agrobacterium tumefaciens. Six plants from 'Pêra' sweet orange and seven plants from 'Valência' sweet orange were confirmed as different transgenic events by means of the polymerase chain reaction (PCR) and the Southern blot techniques. Transgene expression was quantified using real‑time quantitative PCR. D4E1 gene expression levels vary from 5 up to 50 times among different transformation events.

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Rapid Detection of Thymidylate Synthase Gene Expression Levels by Semi-Quantitative Competitive Reverse Transcriptase Polymerase Chain Reaction followed by Quantitative Digital Image Analysis

Tumor Biology ◽

10.1159/000217993 ◽

1996 ◽

Vol 17 (5) ◽

pp. 306-319 ◽

Cited By ~ 4

Author(s):

Thomas A. Houze ◽

Lars Larsson ◽

Per-Anders Larsson ◽

Goran Hansson ◽

Alexzander Asea ◽

...

Keyword(s):

Gene Expression ◽

Polymerase Chain Reaction ◽

Image Analysis ◽

Digital Image ◽

Thymidylate Synthase ◽

Rapid Detection ◽

Digital Image Analysis ◽

Chain Reaction ◽

Polymerase Chain ◽

Gene Expression Levels

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Detection of ERG and MN1 Gene Expression in Adult Patients with De Novo Acute myeloid Leukemia(AML) by Real-Time Quantitative Polymerase Chain Reaction and Its Clinical Significance.

Blood ◽

10.1182/blood.v114.22.4706.4706 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4706-4706

Author(s):

Bing Xu ◽

Xutao Guo ◽

Guoshu Chen ◽

Xiaoyan Song ◽

Pengcheng Shi ◽

...

Keyword(s):

Gene Expression ◽

Polymerase Chain Reaction ◽

Real Time ◽

Clinical Significance ◽

De Novo ◽

Risk Group ◽

Expression Level ◽

Chain Reaction ◽

Expression Levels ◽

Polymerase Chain

Abstract Abstract 4706 Introdution Current study found that certain genetic abnormalities are related to the prognosis of AML, such as ERG and MN1 gene expression. However, little is known about the clinical significance of ERG and MN1 expression level in Chinese patients with AML and and their correlation. Patients and methods A real time quantitative reverse transcriptase polymerase chain reaction method was established for detecting ERG and MN1 gene expression levels in 87 de novo AML patients. Results The median expression levels of ERG and MN1 in AML patient were statistically higher than that in normal control group (P<0.001). ERG gene expression had no correlation with MN1 gene expression (P=0.194) in AML patients. ERG and MN1 gene expressions were equally distributed among the FAB subtypes (P=0.850 and 0.870). Spearman's rank correlation showed that leukocyte counts and lactate dehydrogenase were significantly related to high ERG expression (P=0.005 and 0.032), but not significant correlation was found between hemoglobin and platelet counts. The ERG expression level of cases with middle and low risk group was lower than that of cases with high risk group (P<0.036). There was no significant difference of CR rates in high and low ERG and MN1 expression groups after chemical theropy (P=0.968 and 0.695). During the follow-up of one year, the cumulative relapse rates of high ERG expression groups was significantly higher than that of low ERG expression groups(P=0.039), and high ERG expression cases have a significantly worse OS than low ERG expression cases(P=0.005). Conclusions There isn't a linear correlation between ERG gene expression and MN1 gene expression in AML. Overexpression of ERG gene is a independent prognositic factor for AML. Quantification of the two gene expression together is not more effective to the judgement of prognosis in AML. Disclosures: No relevant conflicts of interest to declare.

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