Statistical analysis of real-time PCR data

BACKGROUND: Even though real-time PCR has been broadly applied in biomedical sciences, data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other useful data analysis methods, we present and compare four statistical approaches and models for the analysis of real-time PCR data. RESULTS: In the first approach, a multiple regression analysis model was developed to derive ΔΔCt from estimation of interaction of gene and treatment effects. In the second approach, an ANCOVA (analysis of covariance) model was proposed, and the ΔΔCt can be derived from analysis of effects of variables. The other two models involve calculation ΔCt followed by a two group t-test and non-parametric analogous Wilcoxon test. SAS programs were developed for all four models and data output for analysis of a sample set are presented. In addition, a data quality control model was developed and implemented using SAS. CONCLUSION: Practical statistical solutions with SAS programs were developed for real-time PCR data and a sample dataset was analyzed with the SAS programs. The analysis using the various models and programs yielded similar results. Data quality control and analysis procedures presented here provide statistical elements for the estimation of the relative expression of genes using real-time PCR

Orexin-A exerts equivocal role in atherosclerosis process depending on the duration of exposure : in vitro study

Orexin-A is a peptide hormone that plays a crucial role in feeding regulation and energy homeostasis. Diurnal intermittent fasting (DIF) has been found to increase orexin-A plasma levels during fasting hours, while Ramadan fasting which resembles DIF, has led to beneficial effects on endothelial function. Herein, we aimed to investigate the effects of orexin-A on the expression of molecules involved in the atherogenesis process: Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), in human aortic endothelial cells (HAECs). HAECs were incubated with orexin-A at concentrations of 40 ng/mL, 200 ng/mL and 400 ng/mL for 6, 12 and 24 h. The mRNA levels of MCP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 and orexin-1 receptor were measured by real-time qPCR. We also evaluated the MMP-2, p38, phospho-p38, NF-κΒ/p65 as well as TIMP-1 protein levels by Western blot and ELISA, respectively. MMP-2 activity was measured by gelatin zymography. Short-term 6-h incubation of HAECs with orexin-A at a high concentration (400 ng/mL) decreased MCP-1, MMP-2 expression, MMP-2/TIMP-1 ratio (p < 0.05), and MMP-2 activity, while incubation for 24 h increased MCP-1, MMP-2 expression (p < 0.05), MMP-2/TIMP-1 and MMP-2/TIMP-2 ratio (p < 0.01 and p < 0.05, respectively) as well as MMP-2 activity. The dual effects of orexin-A are mediated, at least in part, via regulation of p38 and NF-κΒ pathway. Orexin-A may have an equivocal role in atherosclerosis process with its effects depending on the duration of exposure

Evaluating the miR-302b and miR-145 expression in formalin-fixed paraffin-embedded samples of esophageal squamous cell carcinoma

Background: MicroRNAs are involved in key cellular processes regulating, and their misregulation is linked to cancer. The miR-302-367 cluster is exclusively expressed in embryonic stem and carcinoma cells. This cluster also promotes cell reprogramming and stemness process. In contrast, miR-145 is mostly regarded as a tumor suppressor, where it regulates cellular functions such as cell division, differentiation, and apoptosis. By suppressing the main pluripotency factors (OCT4, SOX2, MYC and KLF4), miR-145 silences the self-renewal program in ESCs. Therefore, the main aim of this study is to find a potential link between the expression level of hsa-miR-302b and hsa-miR-145 with tumor vs. non-tumor as well as high-grade vs. low-grade states of the esophageal tissue samples. Methods: A total number of 40 formalin-fixed, paraffin-embedded (FFPE) samples of esophageal squamous-cell carcinoma (ESCC) were obtained, and the tumor and marginal non-tumor areas delineated and punched off by an expert pathologist. Total RNA was extracted with Trizol, and cDNA synthesized using the miRCURY LNA™ Universal RT microRNA PCR Kit. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays were performed using specific LNA-primers and SYBR Green master mix. Results: The expression level of miR-302b failed to show any significant difference, neither between tumor and their non-tumor counterparts, nor among tumors with different grades of malignancies (P > 0.05). In contrast, miR-145 was significantly down regulated in all grades of tumor samples (P 0.05). CONCLUSION: Our data revealed a significant down-regulation of miR-145 in ESCC tissue samples. Based on our ROC curve analysis data (AUC = 0.74, P < 0.001) miR-145 could be regarded as a potential tumor marker for diagnosis of esophageal cancer. © 2015, Academy of Medical Sciences of I.R. Iran. All rights reserved

Expression profile of CREB knockdown in myeloid leukemia cells.

BackgroundThe cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution.MethodsTo understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA.ResultsBy combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA.ConclusionWe have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle

Exploring the genetics of irritable bowel syndrome: A GWA study in the general population and replication in multinational case-control cohorts

OBJECTIVE: IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. DESIGN: We conducted a GWA study (GWAS) of IBS in a general population sample of 11\u2005326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. RESULTS: One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31 710(-6) in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. CONCLUSIONS: Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions

Microbial diversity and biogenic methane potential of a thermogenic-gas coal mine

The microbial communities and biogenic methane potential of a gas coal mine were investigated by cultivation-independent and cultivation-dependent approaches. Stable carbon isotopic analysis indicated that in situ methane in the coal mine was dominantly of a thermogenic origin. However, a high level of diversity of bacteria and methanogens that were present in the coal mine was revealed by 454 pyrosequencing, and included various fermentative bacteria in the phyla of Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria, and acetotrophic, hydrogenotrophic, and methylotrophic methanogens. Methane was produced in enrichments of mine water samples supplemented with acetate under laboratory conditions. The microbial flora obtained from the enrichments could stimulate methane formation from coal samples. 16S rRNA gene clone library analysis indicated that the microbial community from coal cultivation samples supplemented with the enriched microbial consortium was dominated by the anaerobic fermentative Clostridiales and facultative acetoclastic Methanosarcina. This study suggests that the biogenic methane potential in the thermogenic-gas coal mine could be stimulated by the indigenous microorganisms

The Effect of Tumor Microenvironment on Autophagy and Sensitivity to Targeted Therapy in EGFR-Mutated Lung Adenocarcinoma

Lung cancer is the top cancer killer worldwide. Tyrosine kinase inhibitors (TKIs), for example erlotinib, are commonly used to target epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma (ADC). Autophagy is a cellular response to stress, serving as a protective mechanism during anticancer therapy. The tumor microenvironment (TME) is composed of non-tumor cells that include fibroblasts. Our study aimed to investigate the effect of TME on autophagy and TKI sensitivity. Following cell sorting after direct co-culturing, autophagy and cytokine production were observed in both HCC827 and MRC-5 cells. The synergistic combination of erlotinib and chloroquine (autophagy inhibitor) was observed under TME. Tumor growth was significantly suppressed with combined erlotinib/chloroquine compared with erlotinib in HCC827 xenografts.published_or_final_versio

The study of hdac4 gene expression pattern after endurance exercises in animal model

زمینه و هدف: بیشتر تغییرات ایجاد شده در اثر فعالیت های بدنی طی روند اپی ژنیک رخ می دهد که فاکتور رونویسی HDAC4 در این روند نقش محوری بازی می کند. بنابراین هدف این پژوهش بررسی اثر یک دوره فعالیت استقامتی بر بیان ژن hdac4 در عضلات کند و تند انقباض بود. روش بررسی: آزمودنی های این پژوهش تجربی 14 سر موش صحرایی (24&plusmn;231 گرم) بودند که تحت شرایط استاندارد نگهداری شدند. بعد از آشنایی با پروتکل تمرینی به صورت تصادفی به دو گروه کنترل (7=n) و تجربی (7=n) تقسیم شدند. گروه تجربی یک برنامه (30 متر در دقیقه، 50 دقیقه در هر جلسه، 6 جلسه در هفته به مدت 14 هفته) استقامتی را روی تردمیل اجرا کرد و سپس 48 ساعت پس از پایان آخرین جلسه تمرینی همراه با گروه کنترل بی&zwnj;هوش و تشریح شدند، سپس عضله نعلی و عضله EDL آن ها خارج و با استفاده از روش Real time-PCR میزان بیان ژن hdac4 عضلات نعلی و EDL آن ها اندازه&zwnj;گیری شد. در پایان با استفاده از آزمون آماری t میانگین بیان ژن hdac4 گروه تجربی و کنترل ارزیابی و مقایسه شد. یافته ها: نتایج نشان داد، 14 هفته فعالیت استقامتی موجب کاهش بیان ژن hdac4 در عضله نعلی (کند انقباض) می شود به طوری که میزان بیان این ژن در عضله نعلی گروه تجربی به طور معنی داری (0001/0=P) کمتر از گروه کنترل بود. اما در مقایسه با گروه کنترل تفاوت معنی داری (76/0=P) در بیان ژن hdac4 در عضله EDL گروه تجربی مشاهده نشد. نتیجه گیری: این تحقیق نشان داد که علیرغم یکسان بودن شدت و مدت فعالیت استقامتی، بیان ژن hdac4 در عضلات تند و کند انقباض متفاوت بود. کاهش میزان بیان ژن hdac4 در عضله نعلی احتمالاً نشان دهنده رفع فشردگی کروماتین باشد که آغازیست بر بیان ژن&zwnj;های درگیر در فعالیت استقامتی در عضله اسکلتی کند انقباض