Suitable housekeeping genes for normalization of transcript abundance analysis by real-time RT-PCR in cultured bovine granulosa cells during hypoxia and differential cell plating density

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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Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.05132-0 ◽

2003 ◽

Vol 52 (5) ◽

pp. 403-408 ◽

Cited By ~ 205

Author(s):

Hakan Savli ◽

Aynur Karadenizli ◽

Fetiye Kolayli ◽

Sibel Gundes ◽

Ugur Ozbek ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Real Time ◽

Resistance Gene ◽

Housekeeping Genes ◽

Expression Stability ◽

Rt Pcr ◽

Gene Quantification

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Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

JOURNAL FOR CLINICAL AND DIAGNOSTIC RESEARCH ◽

10.7860/jcdr/2021/48726.14977 ◽

2021 ◽

Author(s):

Rajeev Kumar Jain ◽

Nagaraj Perumal ◽

Rakesh Shrivastava ◽

Kamlesh Kumar Ahirwar ◽

Jaya Lalwani ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Internal Control ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Specific Gene ◽

Rt Pcr ◽

Chain Reaction ◽

Gene Target ◽

Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

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Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells

Fertility and Sterility ◽

10.1016/j.fertnstert.2009.01.151 ◽

2010 ◽

Vol 93 (6) ◽

pp. 2050-2055 ◽

Cited By ~ 36

Author(s):

Valério M. Portela ◽

Gustavo Zamberlam ◽

Christopher A. Price

Keyword(s):

Gene Expression ◽

Granulosa Cells ◽

Enzyme Gene ◽

Plating Density ◽

Cell Plating

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Novel porcine housekeeping genes for real-time RT-PCR experiments normalization in adipose tissue: Assessment of leptin mRNA quantity in different pig breeds

Meat Science ◽

10.1016/j.meatsci.2010.10.008 ◽

2011 ◽

Vol 87 (3) ◽

pp. 191-195 ◽

Cited By ~ 16

Author(s):

K. Piórkowska ◽

M. Oczkowicz ◽

M. Różycki ◽

K. Ropka-Molik ◽

A. Piestrzyńska- Kajtoch

Keyword(s):

Adipose Tissue ◽

Real Time ◽

Housekeeping Genes ◽

Rt Pcr ◽

Pig Breeds

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Gonadotropin Stimulation of Ovarian Fractalkine Expression and Fractalkine Augmentation of Progesterone Biosynthesis by Luteinizing Granulosa Cells

Endocrinology ◽

10.1210/en.2007-1662 ◽

2008 ◽

Vol 149 (6) ◽

pp. 2782-2789 ◽

Cited By ~ 16

Author(s):

Ping Zhao ◽

Ananya De ◽

Zeng Hu ◽

Jing Li ◽

Sabine M. Mulders ◽

...

Keyword(s):

Real Time ◽

Granulosa Cells ◽

Regulatory Protein ◽

Rt Pcr ◽

Paracrine Factors ◽

Transcript Levels ◽

Preovulatory Follicles ◽

Major Increase ◽

Steroidogenic Acute Regulatory ◽

Stimulation Of

Recent studies indicated that ovarian functions are regulated by diverse paracrine factors induced by the preovulatory increases in circulating LH. Based on DNA microarray analyses and real-time RT-PCR, we found a major increase in the transcript levels of a chemokine fractalkine after human chorionic gonadotropin (hCG) treatment during the preovulatory period in gonadotropin-primed immature mice and rats. Although CX3CR1, the seven-transmembrane receptor for fractalkine, was also found in murine ovaries, its transcripts displayed minimal changes. Using tandem RT-PCR and immunohistochemistry, fractalkine transcripts and proteins were localized in cumulus, mural granulosa, and theca cells as well as the oocytes, whereas CX3CR1 was found in the same cells except the oocyte. Real-time RT-PCR further indicated the hCG induction of fractalkine transcripts in different ovarian compartments, with the highest increases found in granulosa cells. In cultured granulosa cells, treatment with fractalkine augmented hCG stimulation of progesterone but not estradiol and cAMP biosynthesis with concomitant increases in transcript levels for key steroidogenic enzymes (steroidogenic acute regulatory protein, CYP11A, and 3β-hydroxysteroid dehydrogenase). In cultured preovulatory follicles, treatment with fractalkine also augmented progesterone production stimulated by hCG. Furthermore, treatment with fractalkine augmented the phosphorylation of P38 MAPK in cultured granulosa cells. The present data demonstrated that increases in preovulatory LH/hCG induce the expression of fractalkine to augment the luteinization of preovulatory granulosa cells and suggest the fractalkine/CX3CR1 signaling system plays a potential paracrine/autocrine role in preovulatory follicles.

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Effects of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR

Physiological Genomics ◽

10.1152/physiolgenomics.00060.2002 ◽

2003 ◽

Vol 12 (2) ◽

pp. 163-174 ◽

Cited By ~ 60

Author(s):

R. M. Murphy ◽

K. K. O. Watt ◽

D. Cameron-Smith ◽

C. J. Gibbons ◽

R. J. Snow

Keyword(s):

Skeletal Muscle ◽

Real Time ◽

Human Skeletal Muscle ◽

Detection System ◽

Creatine Supplementation ◽

Housekeeping Genes ◽

Validity And Reliability ◽

Rt Pcr ◽

Study Gene Expression ◽

Total Creatine

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (CT) values were established for β-actin, β2-microglobulin (β2M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between CT values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw CT values and the linear value of 2−CT, respectively. Interassay variability was 2.3% for raw CT values and 34% for the linear value of 2−CT. We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased ( P < 0.05) muscle total creatine content above placebo levels; however, there were no changes ( P > 0.05) in CT values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas β-actin, β2M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for β2M with more stable expressions for both β-actin and CYC. We conclude that, using real-time RT-PCR, β-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise.

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Real-Time Quantitative Reverse Transcription-PCR Analysis of Expression Stability of Actinobacillus pleuropneumoniae Housekeeping Genes during In Vitro Growth under Iron-Depleted Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.71.6.2949-2954.2005 ◽

2005 ◽

Vol 71 (6) ◽

pp. 2949-2954 ◽

Cited By ~ 36

Author(s):

K. Klitgaard Nielsen ◽

M. Boye

Keyword(s):

Gene Expression ◽

Real Time ◽

Reverse Transcription ◽

Geometric Mean ◽

Housekeeping Genes ◽

Actinobacillus Pleuropneumoniae ◽

Internal Controls ◽

Rt Pcr ◽

Bacterial Gene

ABSTRACT The aims of the present investigation were to develop and test a sensitive and reproducible method for the study of gene expression in the porcine lung pathogen Actinobacillus pleuropneumoniae by real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal controls, as such controls have not been defined yet for this bacterium. Bacterial gene expression was studied during in vitro exponential and early stationary growth in medium with and without sufficient iron, respectively. First, the stability of expression of five genes, the glyA, tpiA, pykA, recF, and rhoAP genes involved in basic housekeeping, was evaluated on the basis of the mean pairwise variation. All the housekeeping genes included were stably expressed under the conditions investigated and consequently were included in the normalization procedure. Next, the geometric mean of the internal control genes was used to correct five genes of interest. These genes were three genes involved in iron acquisition (tbpA, exbB, and fhuD), the heat shock protein gene groEL, and a putative quorum-sensing gene (luxS). The level of tbpA, exbB, and fhuD expression in A. pleuropneumoniae showed significant up-regulation under iron-restricted conditions compared to bacteria grown in medium with sufficient iron. The observed expression patterns of the genes of interest were consistent with previous observations. This study therefore lends further support to the use of real-time quantitative RT-PCR, with the glyA, tpiA, pykA, recF, and rhoAP genes as internal controls, for future similar gene expression studies in A. pleuropneumoniae.

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Selection of housekeeping genes for normalization by real-time RT–PCR: Analysis of Or-MYB1 gene expression in Orobanche ramosa development

Analytical Biochemistry ◽

10.1016/j.ab.2008.05.003 ◽

2008 ◽

Vol 379 (2) ◽

pp. 176-181 ◽

Cited By ~ 35

Author(s):

C.I. González-Verdejo ◽

J.V. Die ◽

S. Nadal ◽

A. Jiménez-Marín ◽

M.T. Moreno ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Housekeeping Genes ◽

Pcr Analysis ◽

Rt Pcr ◽

Orobanche Ramosa ◽

Selection Of

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Poly (ADP-ribose) polymerase inhibitor exposure reduces ovarian reserve followed by dysfunction in granulosa cells

Scientific Reports ◽

10.1038/s41598-020-74087-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kentaro Nakamura ◽

Seido Takae ◽

Eriko Shiraishi ◽

Kiemi Shinya ◽

Arby Jane Igualada ◽

...

Keyword(s):

Real Time ◽

Granulosa Cells ◽

Ovarian Function ◽

Histological Study ◽

Parp Inhibitor ◽

Treated Group ◽

Parp Inhibitors ◽

Rt Pcr

Abstract The use of poly (ADP-ribose) polymerase (PARP) inhibitors is expected to increase, but their effect on fertility is still unclear. The aim of this study was to investigate the effect of PARP inhibitors on ovarian function. In an in vitro study, cultures of ovaries and granulosa cells (GCs) exposed to the PARP inhibitor olaparib were evaluated by real-time RT-PCR, histological study, and hormone assays. In an in vivo study, mice were administered olaparib orally and evaluated via in vitro fertilization (IVF), follicle count, immunohistochemical staining, and real-time RT-PCR. In vitro, the gene expression of GC markers decreased in the olaparib-treated group. Olaparib also negatively affected estradiol production and the expression of GC markers in cultured GCs, with abnormal morphology of GCs observed in the treated group. The follicle number indicated depletion of follicles due to atretic changes in the treatment group, both in vitro and in vivo. Also, olaparib reduced the number of retrieved oocytes and the fertilization rate of IVF, but they recovered after 3 weeks of cessation. Our results indicate that olaparib is toxic to ovaries.

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