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

Reproductive Biology and Endocrinology ◽

10.1186/1477-7827-12-118 ◽

2014 ◽

Vol 12 (1) ◽

pp. 118 ◽

Cited By ~ 17

Author(s):

Vijay S Baddela ◽

Anja Baufeld ◽

Vengala R Yenuganti ◽

Jens Vanselow ◽

Dheer Singh

Keyword(s):

Real Time ◽

Granulosa Cells ◽

Housekeeping Genes ◽

Transcript Abundance ◽

Rt Pcr ◽

Plating Density ◽

Differential Cell ◽

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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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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Use of quantitative real-time RT-PCR to analyse the expression of some quorum-sensing regulated genes in Pseudomonas aeruginosa

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-006-0909-0 ◽

2006 ◽

Vol 387 (2) ◽

pp. 513-521 ◽

Cited By ~ 17

Author(s):

Thomas Schwartz ◽

Sandra Walter ◽

Silke-Mareike Marten ◽

Frank Kirschhöfer ◽

Michael Nusser ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Real Time ◽

Rt Pcr

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Reference Gene Validation for Quantitative Real-time PCR Studies in Amphibian Kidney-derived A6 Epithelial Cells

Alternatives to Laboratory Animals ◽

10.1177/0261192919862936 ◽

2019 ◽

Vol 47 (2) ◽

pp. 63-70 ◽

Cited By ~ 2

Author(s):

Elin Verbrugghe ◽

An Martel ◽

Frank Pasmans

Keyword(s):

Cell Line ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Tissue Sample ◽

Housekeeping Genes ◽

Optimal Number ◽

Pairwise Variation ◽

Expression Stability ◽

Specific Tissue

Quantitative real-time polymerase chain reaction is a widely used technique that relies on reference genes for the normalisation of gene expression. These reference genes are constitutively expressed and must remain stable across all samples and treatments. Stability of housekeeping genes may vary and must be optimised for a specific tissue, sample or cell line. Here we present a study screening for possible reference gene candidates, eef1a1, rpl8, sub1.L, clta, H4 and odc1, in the Xenopus laevis (A6) kidney cell line. Quantification cycle results were analysed using geNorm to calculate the average expression stability and the coefficient of variation (CV) for each candidate reference gene. All of the tested genes met the guidelines for stable reference genes, namely an average expression stability of < 0.5 and a CV value of < 0.2, with eef1a1 > sub1.L > rpl8 > clta > odc1 > H4. By using pairwise variation analysis, the optimal number of reference targets was determined to be 2. As such, we report that the reference genes eef1a1 and sub1.L should be used to achieve optimal normalisation in A6 cells.

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