A functional genomic approach to identify reference genes for human pancreatic beta cell real-time quantitative RT-PCR analysis

Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression. We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-BH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples. We identified a total of 264 genes stably expressed in EndoC-BH1 cells and human islets following cytokine- or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.

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Identification and validation of reference genes for real-time quantitative RT-PCR analysis in jute

BMC Molecular Biology ◽

10.1186/s12867-019-0130-2 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 9

Author(s):

Md. Sabbir Hossain ◽

Rasel Ahmed ◽

Md. Samiul Haque ◽

Md. Monjurul Alam ◽

Md. Shahidul Islam

Keyword(s):

Real Time ◽

Reference Genes ◽

Pcr Analysis ◽

Rt Pcr

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Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus)

Gene ◽

10.1016/j.gene.2013.06.013 ◽

2013 ◽

Vol 527 (1) ◽

pp. 183-192 ◽

Cited By ~ 68

Author(s):

Chang Geng Yang ◽

Xian Li Wang ◽

Juan Tian ◽

Wei Liu ◽

Fan Wu ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Oreochromis Niloticus ◽

Reference Genes ◽

Nile Tilapia ◽

Pcr Analysis ◽

Rt Pcr ◽

Nile Tilapia Oreochromis Niloticus

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An optimized protocol for stepwise optimization of real-time RT-PCR analysis

Horticulture Research ◽

10.1038/s41438-021-00616-w ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Fangzhou Zhao ◽

Nathan A. Maren ◽

Pawel Z. Kosentka ◽

Ying-Yu Liao ◽

Hongyan Lu ◽

...

Keyword(s):

Real Time ◽

Reference Genes ◽

Primer Design ◽

Plant Genome ◽

Pcr Analysis ◽

Logarithmic Scale ◽

Rt Pcr ◽

Qpcr Analysis ◽

Specificity And Sensitivity ◽

Optimized Protocol

AbstractComputational tool-assisted primer design for real-time reverse transcription (RT) PCR (qPCR) analysis largely ignores the sequence similarities between sequences of homologous genes in a plant genome. It can lead to false confidence in the quality of the designed primers, which sometimes results in skipping the optimization steps for qPCR. However, the optimization of qPCR parameters plays an essential role in the efficiency, specificity, and sensitivity of each gene’s primers. Here, we proposed an optimized approach to sequentially optimizing primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference (and target) gene. Our approach started with a sequence-specific primer design that should be based on the single-nucleotide polymorphisms (SNPs) present in all the homologous sequences for each of the reference (and target) genes under study. By combining the efficiency calibrated and standard curve methods with the 2−ΔΔCt method, the standard cDNA concentration curve with a logarithmic scale was obtained for each primer pair for each gene. As a result, an R2 ≥ 0.9999 and the efficiency (E) = 100 ± 5% should be achieved for the best primer pair of each gene, which serve as the prerequisite for using the 2−ΔΔCt method for data analysis. We applied our newly developed approach to identify the best reference genes in different tissues and at various inflorescence developmental stages of Tripidium ravennae, an ornamental and biomass grass, and validated their utility under varying abiotic stress conditions. We also applied this approach to test the expression stability of six reference genes in soybean under biotic stress treatment with Xanthomonas axonopodis pv. glycines (Xag). Thus, these case studies demonstrated the effectiveness of our optimized protocol for qPCR analysis.

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rpoB and efp are stable candidate reference genes for quantitative real-time PCR analysis in Saccharopolyspora spinosa

Biotechnology & Biotechnological Equipment ◽

10.1080/13102818.2021.1899852 ◽

2021 ◽

Vol 35 (1) ◽

pp. 619-632

Author(s):

Xiaomeng Liu ◽

Yunpeng Zhang ◽

Kexue Huang ◽

Tie Yin ◽

Qi Li ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Saccharopolyspora Spinosa

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Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) – ITI target genes in mouse ovary identified by microarray analysis

Journal of Endocrinology ◽

10.1677/joe.1.05803 ◽

2004 ◽

Vol 183 (1) ◽

pp. 29-38 ◽

Cited By ~ 14

Author(s):

Mika Suzuki ◽

Hiroshi Kobayashi ◽

Yoshiko Tanaka ◽

Naohiro Kanayama ◽

Toshihiko Terao

Keyword(s):

Real Time ◽

Trypsin Inhibitor ◽

Target Genes ◽

Reproductive Function ◽

Pcr Analysis ◽

Rt Pcr ◽

Reproductive Process ◽

Female Mice ◽

Retinoid Metabolism ◽

Regulatory Effects

Bikunin, a Kunitz-type protease inhibitor, is found in blood and urine. It has been established by two laboratories independently that the bikunin knockout female mice display a severe reduction in fertility: the cumulus oophorus has a defect in forming the extracellular hyaluronan-rich matrix during expansion. Proteins of the inter-alpha-trypsin inhibitor (ITI) family are eliminated in mice in which the bikunin gene has been inactivated, since bikunin is essential for their biosynthesis. Proteins of the ITI family may contribute to the microenvironment in which ovulation takes place. It is not clear, however, whether a single mechanism affects the reproductive function including ovulation. For identifying the full repertoire of the ITI deficiency-related genes, a cDNA microarray hybridization screening was conducted using mRNA from ovaries of wild-type or bik−/− female mice. A number of genes were identified and their regulation was confirmed by real-time RT-PCR analysis. Our screen identified that 29 (0.7%) and 5 genes (0.1%) of the genes assayed were, respectively, up- and down-regulated twofold or more. The identified genes can be classified into distinct subsets. These include stress-related, apoptosis-related, proteases, signaling molecules, aging-related, cytokines, hyaluronan metabolism and signaling, reactive oxygen species-related, and retinoid metabolism, which have previously been implicated in enhancing follicle development and/or ovulation. Real-time RT-PCR analysis confirmed that these genes were up- and down-regulated two- to tenfold by bikunin knockout. These studies demonstrate that proteins of the ITI family may exert potent regulatory effects on a major physiological reproductive process, ovulation.

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Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts

Physiological Genomics ◽

10.1152/physiolgenomics.00004.2012 ◽

2012 ◽

Vol 44 (12) ◽

pp. 651-656 ◽

Cited By ~ 6

Author(s):

S. Ellefsen ◽

M. Bliksøen ◽

A. Rutkovskiy ◽

I. B. Johansen ◽

M.-L. Kaljusto ◽

...

Keyword(s):

Infarct Size ◽

Real Time ◽

Reference Genes ◽

Stable Expression ◽

Unit Weight ◽

Living Tissue ◽

Rt Pcr ◽

Internal Reference ◽

Total Rna ◽

Rat Hearts

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability ( R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, β-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1–50.1%). When β-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.

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Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise

Physiological Genomics ◽

10.1152/physiolgenomics.00067.2004 ◽

2004 ◽

Vol 18 (2) ◽

pp. 226-231 ◽

Cited By ~ 144

Author(s):

Douglas J. Mahoney ◽

Kate Carey ◽

Ming-Hua Fu ◽

Rodney Snow ◽

David Cameron-Smith ◽

...

Keyword(s):

Skeletal Muscle ◽

Real Time ◽

Healthy Subjects ◽

Acute Exercise ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Cycle Ergometry ◽

Pcr Analysis ◽

28S Rrna ◽

Rt Pcr

Studies examining gene expression with RT-PCR typically normalize their mRNA data to a constitutively expressed housekeeping gene. The validity of a particular housekeeping gene must be determined for each experimental intervention. We examined the expression of various housekeeping genes following an acute bout of endurance (END) or resistance (RES) exercise. Twenty-four healthy subjects performed either a interval-type cycle ergometry workout to exhaustion (∼75 min; END) or 300 single-leg eccentric contractions (RES). Muscle biopsies were taken before exercise and 3 h and 48 h following exercise. Real-time RT-PCR was performed on β-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β2-microglobulin (β2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at ∼65% of V̇o2 max, and we examined a fifth housekeeping gene, 28S rRNA, and reexamined β2M, from muscle biopsy samples taken immediately postexercise. We showed that CYC increased 48 h following both END and RES exercise (3- and 5-fold, respectively; P < 0.01), and 28S rRNA increased immediately following END exercise (2-fold; P = 0.02). β-Actin trended toward an increase following END exercise (1.85-fold collapsed across time; P = 0.13), and GAPDH trended toward a small yet robust increase at 3 h following RES exercise (1.4-fold; P = 0.067). In contrast, β2M was not altered at any time point postexercise. We conclude that β2M and β-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas β2M and GAPDH are the most stably expressed following END exercise.

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Pioglitazone Attenuates Vascular Fibrosis in Spontaneously Hypertensive Rats

PPAR Research ◽

10.1155/2012/856426 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Dengfeng Gao ◽

Ning Ning ◽

Guanghua Hao ◽

Xiaolin Niu

Keyword(s):

Real Time ◽

Immunohistochemical Staining ◽

Spontaneously Hypertensive Rats ◽

Pcr Analysis ◽

Rt Pcr ◽

Hypertensive Rats ◽

Spontaneously Hypertensive ◽

Ppar Γ ◽

Collagen Iii ◽

Wistar Kyoto

Objective. We sought to investigate whether the peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand pioglitazone can attenuate vascular fibrosis in spontaneously hypertensive rats (SHRs) and explore the possible molecular mechanisms.Methods. SHRs (8-week-old males) were randomly divided into 3 groups (n=8each) for treatment: pioglitazone (10 mg/kg/day), hydralazine (25 mg/kg/day), or saline. Normal male Wistar Kyoto (WKY) rats (n=8) served as normal controls. Twelve weeks later, we evaluated the effect of pioglitazone on vascular fibrosis by Masson’s trichrome and immunohistochemical staining of collagen III and real-time RT-PCR analysis of collagen I, III and fibronectin mRNA.Vascular expression of PPAR-γ and connective tissue growth factor (CTGF) and transforming growth factor-β (TGF-β) expression were evaluated by immunohistochemical staining, western blot analysis, and real-time RT-PCR.Results. Pioglitazone and hydralazine treatment significantly decreased systolic blood pressure in SHRs. Masson’s trichrome staining for collagen III and real-time RT-PCR analysis of collagen I, III and fibronectin mRNA indicated that pioglitazone significantly inhibited extracellular matrix production in the aorta. Compared with Wistar Kyoto rats, SHRs showed significantly increased vascular CTGF expression. Pioglitazone treatment significantly increased PPAR-γ expression and inhibited CTGF expression but had no effect on TGF-β expression.Conclusions. The results indicate that pioglitazone attenuated vascular fibrosis in SHRs by inhibiting CTGF expression in a TGF-β-independent mechanism.

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