Stable Endogenous Control Research Articles

MiR-122, miR-133a, and miR-206 as potential biomarkers for post-mortem interval estimation.

Accurate estimation of post-mortem interval (PMI) is crucial in forensic investigations. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that remain relatively stable within the cell nucleus despite post-mortem changes. We assessed three target genes (miR-122, miR-133a, and miR-206) for PMI estimation using 72 healthy adult male BALB/c mice exposed to two different temperatures (4 and 21℃) at nine different time points over 10days. Initially, the stability of the two reference genes (RNU6B and 5 srRNA) was evaluated using gene stability analysis tools (Delta Ct, Best Keeper, and Genorm) to select the optimal reference gene. RNU6B was found to be the most stable endogenous control. Subsequently, the expression patterns of miR-122, miR-133a, and miR-206 were analyzed within a 10-day PMI period using the heart, skeletal muscle, liver, and brain tissues. At 4℃, miR-122 levels significantly decreased on days 8 and 10 in all tissues, with only the liver showing significant changes at 21℃. MiR-133a decreased over time in the heart, muscles, and brain, showing a dramatic decrease on days 8 and 10 in the heart and muscles at both temperatures. Although miR-206 levels decreased over time in muscles and liver at 4℃, these increased in the brain at 21℃, with no expression changes in other organs. In summary, miR-122, miR-133a, and miR-206 are potential PMI markers in heart and skeletal muscle tissues.

Abstract 2350: Comparison of normalization methods for RT-qPCR microRNA expression in cancer datasets

Abstract Real-time quantitative PCR (RT-qPCR) is a widely used method for quantifying microRNA (miRNA) expression, but its accuracy depends on appropriate normalization. RT-qPCR is susceptible to technical variation from several sources, including sample collection and storage, miRNA quantity/quality, and extraction and amplification efficiencies. Normalization corrects for these factors, theoretically leaving behind only biological variation. With growing interest in miRNAs as biomarkers for the diagnosis and prognosis of various cancers, analyses must be accurate and consistent. Primarily, the comparative Ct method is used, which compares the expression of miRNAs to a reference value. Unfortunately, there is a lack of consistency on what to use as a reference. A standard recommendation is the average of multiple, stable miRNAs referred to as endogenous controls (ECs). Currently, there are no known universally stable miRNAs. Therefore, ECs should be selected on a per-disease and per-sample type basis. Multiple algorithms exist to identify stable ECs, but few studies indicate what, if any, steps were taken to implement them correctly. Also, many studies have used non-ideal reference values such as small nuclear RNAs or a single miRNA. This lack of agreement on protocol often leads to difficulty comparing studies and may produce incorrect results. This work addresses these inconsistencies by making recommendations for the normalization of RT-qPCR miRNA expression. We compared 2 widely used methods for EC selection, NormFinder and GeNorm, and to show how normalization influences results we also used an unstable miRNA. Only miRNAs with expression in all samples were considered as potential ECs. GeNorm is sensitive to correlated genes. Therefore, for any pair or trio of correlated miRNAs 1 was kept. NormFinder performs optimally with 5-10 candidate genes; therefore, we tested the 10 miRNAs with the lowest coefficients of variation (CV). The top 3 stable miRNAs were selected from each algorithm to serve as ECs, and we used their average expression to normalize each sample. For the single miRNA, samples were scaled based on its value. We applied these methods to 3 independent datasets: First, plasma samples from a study on canine osteosarcoma (OSA), including pre-amputation (n=45), post-amputation (n=27), and healthy controls (n=21). Second, tissue samples from a study on canine OSA, including primary tumour (n=42) and lung metastases (n=12). Third, serum samples from a study on canine lymphoma included B-cell (n=24), T-cell (n=16), and healthy controls (n=14). For datasets 1 and 3, NormFinder provided a better reduction in gene-specific CV and a more favourable cumulative distribution of the CV. For dataset 2, there was less initial variability, but NormFinder still had slightly better results. Both algorithms had pros and cons, but NormFinder consistently provides a better reduction in sample variation across datasets. Citation Format: Heather Treleaven, Latasha Ludwig, Alicia Viloria-Petit, R. Darren Wood, Ayesha Ali, Geoffrey A. Wood. Comparison of normalization methods for RT-qPCR microRNA expression in cancer datasets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2350.

Identification of stably expressed microRNAs in plasma from high-grade serous ovarian carcinoma and benign tumor patients

BackgroundOvarian cancer is a lethal gynecological cancer and no reliable minimally invasive early diagnosis tools exist. High grade serous ovarian carcinoma (HGSOC) is often diagnosed at advanced stages, resulting in poorer outcome than those diagnosed in early stage. Circulating microRNAs have been investigated for their biomarker potential. However, due to lack of standardization methods for microRNA detection, there is no consensus, which microRNAs should be used as stable endogenous controls. We aimed to identify microRNAs that are stably expressed in plasma of HGSOC and benign ovarian tumor patients.Methods and resultsWe isolated RNA from plasma samples of 60 HGSOC and 48 benign patients. RT-qPCR was accomplished with a custom panel covering 40 microRNAs and 8 controls. Stability analysis was performed using five algorithms: Normfinder, geNorm, Delta-Ct, BestKeeper and RefFinder using an R-package; RefSeeker developed by our study group [1]. Among 41 analyzed RNAs, 13 were present in all samples and eligible for stability analysis. Differences between stability rankings were observed across algorithms. In HGSOC samples, hsa-miR-126-3p and hsa-miR-23a-3p were identified as the two most stable miRNAs. In benign samples, hsa-miR-191-5p and hsa-miR-27a-3p were most stable. In the combined HGSOC and benign group, hsa-miR-23a-3p and hsa-miR-27a-3p were identified by both the RefFinder and Normfinder analysis as the most stable miRNAs.ConclusionsConsensus regarding normalization approaches in microRNA studies is needed. The choice of endogenous microRNAs used for normalization depends on the histological content of the cohort. Furthermore, normalization also depends on the algorithms used for stability analysis.

Strategies for data normalization and missing data imputation and consequences for potential diagnostic microRNA biomarkers in epithelial ovarian cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression with diagnostic potential in different diseases, including epithelial ovarian carcinomas (EOC). As only a few studies have been published on the identification of stable endogenous miRNA in EOC, there is no consensus which miRNAs should be used aiming standardization. Currently, U6-snRNA is widely adopted as a normalization control in RT-qPCR when investigating miRNAs in EOC; despite its variable expression across cancers being reported. Therefore, our goal was to compare different missing data and normalization approaches to investigate their impact on the choice of stable endogenous controls and subsequent survival analysis while performing expression analysis of miRNAs by RT-qPCR in most frequent subtype of EOC: high-grade serous carcinoma (HGSC). 40 miRNAs were included based on their potential as stable endogenous controls or as biomarkers in EOC. Following RNA extraction from formalin-fixed paraffin embedded tissues from 63 HGSC patients, RT-qPCR was performed with a custom panel covering 40 target miRNAs and 8 controls. The raw data was analyzed by applying various strategies regarding choosing stable endogenous controls (geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder), missing data (single/multiple imputation), and normalization (endogenous miRNA controls, U6-snRNA or global mean). Based on our study, we propose hsa-miR-23a-3p and hsa-miR-193a-5p, but not U6-snRNA as endogenous controls in HGSC patients. Our findings are validated in two external cohorts retrieved from the NCBI Gene Expression Omnibus database. We present that the outcome of stability analysis depends on the histological composition of the cohort, and it might suggest unique pattern of miRNA stability profiles for each subtype of EOC. Moreover, our data demonstrates the challenge of miRNA data analysis by presenting various outcomes from normalization and missing data imputation strategies on survival analysis.

MiR-423-5p is a novel endogenous control for the quantification of circulating miRNAs in human esophageal squamous cell carcinoma

Circulating miRNA expression is most commonly measured by qRT-PCR, however, the lack of a suitable endogenous control hinders people from evaluating the accurate changes in miRNA expression levels and developing the non-invasive biomarkers. In this study, we aimed to screen the specific, highly stable endogenous control in esophageal squamous cell carcinoma (ESCC) to overcome the obstacle. We selected “housekeeping” miRNAs according to the published database and initially acquired 21 miRNAs. Subsequently, we screened these miRNAs using GSE106817 and TCGA datasets according to specific inclusion criteria and evaluated the suitability of “candidate” miRNAs. Among these miRNAs, the average abundance of miR-423-5p was relatively high in serum. Notably, miR-423-5p expression in serum showed no significant difference between ESCC patients and healthy controls (n = 188, P = 0.29). Moreover, among these miRNAs, miR-423-5p was the most stable miRNA using the NormFinder algorithms. Overall, these results indicate that miR-423-5p, as a novel and optimal endogenous control, could be used to quantify circulating miRNAs in ESCC.

Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions

Cell-free miRNAs, called circulating miRNAs (cmiRNAs), can act in a paracrine manner by facilitating a diversity of signaling mechanisms between cells. Real-time qPCR is the most accepted method for quantifying miRNA expression levels. The use of stable miRNA endogenous control (EC) for qPCR data normalization allows an accurate cross-sample gene expression comparison. The appropriate selection of EC is a crucial step because qPCR data can change drastically when normalization is performed using an unstable versus a stable EC. To find EC cmiRNA with stable expression in search and rescue (SAR) working dogs, we explored the serum miRNome by Next-Generation Sequencing (NGS) at T0 (resting state) and T1 immediately after SAR performance (state of physiologically recovered stress). The cmiRNAs selected in the NGS circulating miRNome as probable ECs were validated by qPCR, and miRNA stability was evaluated using the Delta Ct, BestKeeper, NormFinder, and GeNorm algorithms. Finally, RefFinder was used to rank the stability orders at both T0 and T1 by establishing miR-320 and miR-191 as the best-circulating ECs. We are confident that this study not only provides a helpful result in itself but also an experimental design for selecting the best endogenous controls to normalize gene expression for genes beyond circulating miRNAs.

Identification of reference genes for quantitative expression analysis in Indian catfish,Clarias magur, under physiological and pathological conditions

The reverse transcription quantitative real-time PCR (RT-qPCR) is widely preferred tool for expression analysis of target gene at mRNA level. However, in order to obtain significant biological comparison, data normalization to a stable endogenous internal control gene also called reference gene (RG) is of utmost importance. In the present study, we evaluated the expression stability of four commonly used housekeeping genes, that is beta actin, β-actin; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; elongation factor-1 alpha, EF-1α and 18S ribosomal RNA, 18S for qPCR data normalization. This study was conducted for two varying physiological (Group I: fed vs. non-fed and Group II: earthen pond vs. fibre reinforced plastic tank reared) and one pathological condition (Group III: Aeromonas hydrophila injected vs. PBS injected) in seven different tissues of Indian catfish, Clarias magur, an economically valuable food fish of South-East Asia. Tissue wise expression stability analysis using web-based comprehensive tool Reffinder revealed that EF-1α was the most stable RG in kidney, liver, spleen and gills of group I; spleen, intestine, muscle and brain in group II; and kidney, liver, intestine, muscle and brain in group III. β-actin was the most stable RG in intestine of group I; liver and brain in group II; spleen and gill in group III. 18S was the most stable RG in muscle and brain of group I; kidney and gill in group II. GAPDH was the least stable RG among four selected genes under all the experimental conditions. These findings on selection of RGs will lead to precise interpretation of relative gene expression in different tissues by RT-qPCR in C. magur in the context of physiological studies and bacterial infection.

Identification of Stably Expressed Reference microRNAs in Epithelial Ovarian Cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression and have been associated with the development of various cancers, including epithelial ovarian cancer (EOC). Accurate quantification of miRNA levels is important for determining their role in tumorigenesis and as biomarkers. Currently, U6 is widely used as a normalization control when investigating miRNAs in EOC; however, its variable expression across cancers has been reported. As only a few studies have been published to date on the identification of endogenous miRNA controls in EOC, our aim was to identify stable miRNAs based on global microarray profiling of 197 EOC patients and verify their stability in external datasets. We collected miRNA-microarray data from four datasets: the in-house "Pelvic Mass", and three public datasets with primary EOC patients: The Cancer Genome Atlas, GSE47841, and GSE73581. The expression stability of endogenous control candidates was evaluated by their coefficient of variation. All miRNA results in the used cohorts were produced by either Affymetrix or Agilent technologies, which show similar intra-platform patterns. Nonetheless, a clear difference in a cross-platform comparison was observed. We identified hsa-miR-92b-5p and hsa-miR-106b-3p as stable candidates shared between four datasets. Moreover, we investigated the stability performance of eight miRNAs that have been previously reported as stable endogenous controls in EOC and various performance was observed in four datasets. The selection of suitable endogenous miRNA normalization controls in EOC remains to be resolved, as variability in miRNA performance between platforms might have a crucial impact on the biological interpretation of data.

Identification of Circulating miR-22-3p and miR-93-5p as Stable Endogenous Control in Tuberculosis Study.

The diagnosis and prognosis of tuberculosis remains challenging and necessitates the development of a new test that can accurately diagnose and monitor treatment responses. In this regard, miRNA is becoming a potential diagnostic and prognostic biomarker which differentiates treatment respondents from non-respondents for various non-infectious and infectious diseases, including tuberculosis. The concentration of miRNAs varies based on cell type, disease, and site of infection, implicating that selection of an optimal reference gene is crucial, and determines the quantification of transcript level and biological interpretation of the data. Thus, the study evaluated the stability and expression level of five candidate miRNAs (let-7i-5p, let-7a-5p, miRNA-16-5p, miRNA-22-3p and miRNA-93-5p), including U6 Small Nuclear RNA (RNU6B) to normalize circulating miRNAs in the plasma of 68 participants (26 healthy controls, 23 latent, and 19 pulmonary tuberculosis infected) recruited from four health centers and three hospitals in Addis Ababa, Ethiopia. The expression levels of miRNAs isolated from plasma of culture confirmed newly diagnosed pulmonary tuberculosis patients were compared with latently infected and non-infected healthy controls. The qPCR data were analyzed using four independent statistical tools: Best Keeper, Genorm, Normfinder and comparative delta-Ct methods, and the data showed that miRNA-22-3p and miRNA-93-5p were suitable plasma reference miRNAs in a tuberculosis study.

Selection of the Most Stable Endogenous Control Genes for Microrna Quantitation in Chicken Ovarian Follicles

Abstract MicroRNAs (miRNAs or miRs) belong to a class of small non-coding RNAs of 19 to 24 nucleotides long that act as negative gene regulators at the post-transcriptional level. Quantitative PCR (q-PCR) is a commonly used technique in the profiling of miRs, and identification of reliable endogenous controls is crucial for proper data normalisation. To date, no study has been performed on reference miRs for the normalisation of miR expression in chicken ovarian tissues. Therefore, the aim of the present study was to experimentally identify the most stable reference mirs for normalisation of miR q-PCR expression data in the chicken ovary. Relying on high-throughput sequencing, five putative reference miR (let-7a-3p, miR-140a-3p, miR-22-5p, miR-33-5p, miR-99a-3p) were identified and subsequently analysed in a total of 66 tissue samples. The stability of candidate endogenous controls validated by the most widely used algorithms, geNorm, NormFinder, and BestKeeper, showed that let-7a-3p, miR-140a-3p, and miR-22-5p are the most appropriate choice of reference genes. Application of different normalisation approaches to the relative quantitation of randomly chosen miR-1552-5p in chicken ovarian follicles indicated the impact of the selected reference genes on miR expression. Further, the results revealed a downregulation of miR-1552-5p. In summary, the three identified endogenous reference miRs are suitable for profiling the miR expression in ovarian tissues of laying hens. Our findings provide valuable information for future miR expression studies in the avian ovary.

The potential role of miR-126, miR-21 and miR-10b as prognostic biomarkers in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most commonly diagnosed renal tumor, consisting of ~3% of all malignancies worldwide. The prognosis of RCC can vary widely, and detecting patients at risk of recurrence at an early stage of disease may improve patient outcome. The factors presently used in a clinical setting cannot reliably predict the natural history of the disease. Therefore, there is a requirement to identify novel biomarkers that can aid in predicting patient outcome. Previous studies have indicated that microRNAs (miRNAs/miRs) are potential candidates as prognostic biomarkers for patients suffering from RCC. Consequently, the aims of the present study were to validate the potential of 3 of these miRNAs to predict the prognosis of patients with RCC, and to investigate the stability of endogenous control genes for miRNA studies in RCC tissues. The expression of 7 endogenous controls was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in formalin-fixed paraffin-embedded tumor and benign tissues from patients suffering from clear cell RCC (ccRCC). The analyses identified RNU48 and U47 as the most stable endogenous controls. The expression of miR-126, miR-21 and miR-10b was analyzed using RT-qPCR in renal tissues from 116 patients diagnosed with ccRCC. All three investigated miRNAs were differentially expressed between malignant and benign tissues. miR-126 and miR-10b were also differentially expressed between grades and stages of ccRCC. In a univariate, but not in a multivariate model, low expression of miR-126 was associated with shorter time to recurrence of the disease. The results of the present study indicate that of the 3 miRNAs investigated, the expression of miR-126 has the strongest potential as a prognostic biomarker for patients suffering from ccRCC.

Technical note: Evaluation of endogenous control gene expression in bovine neutrophils by reverse-transcription quantitative PCR using microfluidics gene expression arrays.

Reverse-transcription quantitative-PCR (RT-qPCR) is commonly used for assessing the cellular response to changes in physiologic and pathologic conditions. The selection of stable endogenous control genes is an important step of any RT-qPCR study, as expression can vary depending on the experimental environment. Our objective was to identify endogenous control genes in circulating neutrophils isolated from cows during the peripartum period. To do this, we used microfluidics gene expression arrays (Fluidigm, San Francisco, CA) for RT-qPCR analysis. Selection of the endogenous control genes was based on previous research investigating gene expression in neutrophils. The selected genes included ACTB, B2M, G6PD, GAPDH, GCH1, GOLGA5, OSBPL2, PGK1, RPL13A, RPL19, RPS9, SDHA, SMUG1, SNRPA, TBP, UXT, and YWHAZ. Four genes (GAPDH, GOLGA5, PGK1, and UXT) did not provide satisfactory quantification results using the selected method and were therefore excluded from the analyses. The suitability of the remaining 13 genes for use as endogenous control genes was assessed using geNorm and Normfinder. The gene pair with the greatest stability using geNorm was RPL13A and RPL19, whereas Normfinder ranked RPL19 and YWHAZ as the most stable pair. The 2 genes deemed most suitable for the experimental design were RPL19 and YWHAZ, which were selected for subsequent gene expression analysis. This study highlights that genes used as endogenous controls for relative quantification should be assessed on an experimental basis, even if the genes have been used in previous research.

Circulating microRNAs as novel biomarkers of ALK-positive nonsmall cell lung cancer and predictors of response to crizotinib therapy.

Circulating microRNAs are potential diagnostic and predictive biomarkers, but have not been investigated for patients with anaplastic lymphoma kinase (ALK)-positive lung cancer. In this exploratory study, we sought to identify potential plasma biomarkers for ALK-positive non-small cell lung cancer (NSCLC). A microRNA microarray was used to select ALK-related microRNAs in ALK-positive NSCLC (n = 3), ALK-negative NSCLC (n = 3), and healthy subjects (n = 3). Plasma levels of 21 microRNAs were differentially expressed for ALK-positive and ALK-negative NSCLC, including 14 down-regulated and 7 up-regulated microRNAs. We also identified 5s rRNA as the most stable endogenous control gene using geNorm and NormFinder algorithms. Candidate microRNAs in plasma from ALK-positive (n = 41) and ALK-negative NSCLC patients (n = 32) were quantified using real-time reverse transcriptase quantitative polymerase chain reaction. The expression levels of miR-28-5p, miR-362-5p, and miR-660-5p were all down-regulated in ALK-positive NSCLC, compared with ALK-negative NSCLC. The areas under the receiver operating characteristic curves of miR-28-5p, miR-362-5p, miR-660-5p, and 3-microRNAs panel were 0.873, 0.673, 0.760, and 0.876, respectively. The positive predictive values of miR-28-5p, miR-362-5p, and miR-660-5p were 96.43%, 80.77%, and 83.87%, respectively. Increased plasma levels of miR-660-5p after crizotinib treatment predicted good tumor response (p = 0.012). The pre-crizotinib levels of miR-362-5p were significantly associated with progression-free survival (p = 0.015). Thus, in this preliminary investigation, we identified a potential panel of 3 microRNAs for distinguishing between patients with ALK-positive and ALK-negative NSCLC. We also identified miR-660-5p and miR-362-5p as potential predictors for response to crizotinib treatment.

Identification and validation of microRNAs as endogenous controls for quantitative polymerase chain reaction in plasma for stable coronary artery disease.

Circulating microRNAs (miRNAs) have been proved to serve as biomarkers for diagnosis and assessment of prognosis of coronary artery disease (CAD). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a widely-used technique to estimate expression levels of circulating miRNAs. Selection of optimal endogenous control (EC) remains critical to obtain reliable qPCR data of miRNAs expression. However, reference controls for normalization of circulating miRNA in CAD are still lacking. The purpose of this study was to identify stably expressed miRNAs to normalize RT-qPCR data derived from plasma in stable CAD. We identified 10 stably expressed candidate ECs by combining miRNA microarray screening and literature screening. These 10 candidate ECs were estimated by RT-qPCR and the data were analyzed by NormFinder and BestKeeper algorithm. Two most stable ECs were identified as EC candidates and they were subsequently validated in another larger cohort. The 2 candidates were also validated by normalizing the expression levels of miR-21. In general, they were superior to the commonly used reference gene RNU6 in quantification cycle (Cq) value, stability value and normalization effect. Our results demonstrated that miR-6090 and miR-4516 can be used as reference genes for plasma miRNA analysis in stable CAD.

Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum.

Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

The expression of the SCD1 gene and its correlation with fattening and carcass traits in sheep

Abstract. Stearoyl-CoA desaturase 1 (SCD1) is a critical enzyme that catalyzes the synthesis of monounsaturated fatty acids and is involved in several signaling pathways related to lipid metabolism. The objective of the present study was to estimate the expression of the SCD1 gene in three different ovine tissues strongly associated with lipid homeostasis. The SCD1 gene expression measurement was performed on three tissues (liver, subcutaneous fat, perirenal fat) originated from 15 old-type Polish Merino sheep. The SCD1 transcript abundance was evaluated based on the two most stable endogenous controls (RPS2 – ribosomal protein S2; ATP5G2 – H(+)-transporting ATP synthase). The highest expression of the SCD1 gene was observed in ovine subcutaneous fat compared to perirenal fat and liver. Furthermore, the present research indicated the significant correlation between ovine SCD1 transcript abundance and several important production traits. The expression of the SCD1 gene in liver and perirenal fat highly positively correlated with the feed : gain ratio, test of daily gain and age of the animals at slaughter. Moreover, in both tissues, the SCD1mRNA level positively correlated with weight and content of perirenal fat and subcutaneous fat (R = 0.64, 0.8, 0.6, respectively) and negatively with assessment of external fat content with the use of the EUROP scale (R = −0.64). The SCD1 expression in subcutaneous fat also corresponds with back fat of blade chop and thickness of longissimus dorsi muscles evaluated using USG (ultrasonography) (R = −0.6 and 0.62, respectively). The significant correlation between SCD1 transcript abundance and fattening and slaughtering traits indicate the ability to improve important production traits in sheep via modification of expression of the SCD1 gene.

Identification of Ubiquitin C as the most stable endogenous control for gene expression studies in mouse placenta

Identification of Ubiquitin C as the most stable endogenous control for gene expression studies in mouse placenta

Identification of suitable endogenous controls for gene and miRNA expression studies in irradiated prostate cancer cells.

This study aimed to to evaluate the stability of commonly used endogenous control genes for messenger RNA (mRNA) (N = 16) and miRNAs (N = 3) expression studies in prostate cell lines following irradiation. The stability of endogenous control genes expression in irradiated (6 Gy) versus unirradiated controls was quantified using NormFinder and coefficient of variation analyses. HPRT1 and 18S were identified as most and least stable endogenous controls, respectively, for mRNA expression studies in irradiated prostate cells. SNORD48 and miR16 miRNA endogenous controls tested were associated with low coefficient of variations following irradiation (6 Gy). This study highlights that commonly used endogenous controls can be responsive to radiation and validation is required prior to gene/miRNAs expression studies.

The use of circulating microRNAs as diagnostic biomarkers in colorectal cancer

Abnormal levels of microRNAs (miRNAs) have been found in the blood or its components in a number of different cancers including colorectal cancer. In addition to being abundant in circulation, miRNAs show remarkable stability in both plasma and serum making miRNAs ideal markers for early detection in colorectal cancer. Several miRNAs have been identified as potential circulating biomarkers although none have been incorporated into clinical practice. To identify the most consistently dysregulated circulating miRNAs in colorectal cancer patients according to current literature and postulate reasons for heterogeneity in results. A literature review was performed using the electronic databases PubMed, Embase and the Cochrane Library. The 6 circulating miRNAs most frequently found to be dysregulated in colorectal cancer are miR-18a-5p, miR-21-5p, miR-29a-5p, miR-92a-5p, miR-143-5p and miR-378-5p. There are, however, multiple studies with conflicting findings. Studies vary significantly in ethnicity of populations, use of endogenous controls, source of miRNAs (whole blood, serum and plasma) and methods of detection. Circulating miRNAs are promising diagnostic biomarkers in colorectal cancer. Further studies identifying the source of tumour derived miRNAs in circulation, including identification of exosomal miRNA content, are required. Identifying pre-profiling factors affecting miRNA expression and determining stable endogenous controls will expedite the incorporation of miRNAs into clinical practice.

Reference gene selection for qPCR is dependent on cell type rather than treatment in colonic and vaginal human epithelial cell lines.

The ability of commensal bacteria to influence gene expression in host cells under the influence of pathogenic bacteria has previously been demonstrated, however the extent of this interaction is important for understanding how bacteria can be used as probiotics. Real-time quantitative polymerase chain reaction is the most sensitive tool for evaluating relative changes to gene expression levels. However as a result of its sensitivity an appropriate method of normalisation should be used to account for any variation incurred in preparatory experimental procedures. These variations may result from differences in the amount of starting material, quality of extracted RNA, or in the efficiency of the reverse transcriptase or polymerase enzymes. Selection of an endogenous control gene is the preferred method of normalisation, and ideally a proper validation of the gene's appropriateness for the study in question should be performed. In this study we used quantitative polymerase chain reaction data and applied four different algorithms (geNorm, BestKeeper, NormFinder, and comparative ΔCq) to evaluate eleven different genes as to their suitability as endogenous controls for use in studies involving colonic (HT-29) and vaginal (VK2/E6E7) human mucosal epithelial cells treated with probiotic and pathogenic bacteria. We found phosphoglycerate kinase 1 to be most appropriate for HT-29 cells, and ribosomal protein large P0 to be the best choice for VK2/E6E7 cells. We also showed that use of less stable reference genes can lead to less accurate quantification of expression levels of gene of interest (GOI) and also can result in decreased statistical significance for GOI expression levels when compared to control. Additionally, we found the cell type being analysed had greater influence on reference gene selection than the treatment performed. This study provides recommendations for stable endogenous control genes for use in further studies involving colonic and vaginal cell lines after bacterial challenge.