Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR

Schmittgen, Thomas D.; Zakrajsek, Brian A.

doi:10.1016/s0165-022x(00)00129-9

Cited by 1,090 publications

(742 citation statements)

References 5 publications

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“…Overall, our study demonstrates that UBC, YW-HAZ, RPLP and TBP, were the most suitable reference genes to be used for gene expression studies using MCF7, HCT116 and HepG2 cells. Although GAPDH and ACTB are used as reference genes in most in vitro studies using these cell lines (Bracke et al 1993;Tanic et al 2007;Cicinnati et al 2008;Mori et al 2008), these two reference genes did not exhibit stable expression levels across MCF7, HCT116 and HepG2 cells, as demonstrated by our current results as well as previous studies (Schmittgen and Zakrajsek 2000;Gorzelniak et al 2001). The GAPDH and ACTB genes are variably expressed when subjected to serumstimulation (Schmittgen and Zakrajsek 2000) and hormone treatments (Gorzelniak et al 2001).…”

Section: Discussionsupporting

confidence: 73%

“…Although GAPDH and ACTB are used as reference genes in most in vitro studies using these cell lines (Bracke et al 1993;Tanic et al 2007;Cicinnati et al 2008;Mori et al 2008), these two reference genes did not exhibit stable expression levels across MCF7, HCT116 and HepG2 cells, as demonstrated by our current results as well as previous studies (Schmittgen and Zakrajsek 2000;Gorzelniak et al 2001). The GAPDH and ACTB genes are variably expressed when subjected to serumstimulation (Schmittgen and Zakrajsek 2000) and hormone treatments (Gorzelniak et al 2001). The expression of GAPDH is also regulated differently in various human cancer tissues such as breast cancer, prostate cancer, pancreatic adenocarcinoma and bladder cancer (Rondinelli et al 1997;Schek et al 1988;Revillion et al 2000;Ohl et al 2006).…”

Section: Discussionsupporting

confidence: 73%

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UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines

et al. 2011

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Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of C t values. The geNorm analysis showed the following ranking for stability of genes:A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionsupporting

confidence: 73%

UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines

et al. 2011

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“…Glyceraldehyde-3-phosphate-dehydrogenase is an abundant glycolytic enzyme and therefore an increase in transcription is consistent with a higher demand of cellular energy associated with growth and cell differentiation. Normalisation to GAPDH was already reported as valid (Wall and Edwards 2002), however in most cases this gene proved to be inappropriate as endogenous control in quantification assays (Schmittgen and Zakrajsek 2000;Goidin et al 2001;Kim et al 2003). A maximum variability of GAPDH of 25 fold has been reported (Dheda et al 2004), and according to Bustin (2002) for most experimental conditions the use of GAPDH is inappropriate and should be discontinued.…”

Section: Discussionmentioning

confidence: 99%

“…In fact, 18S ribosomal RNA has been reported as a reliable reference gene for real-time RT-PCR in distinct experimental conditions (Schmittgen and Zakrajsek 2000;Goidin et al 2001;Kim et al 2003). However, according to Solanas et al (2001) there are some concerns regarding the use of rRNA as internal standard due to variations in rRNA expression levels, transcription by different RNA polymerases and possible imbalances in rRNA and mRNA fractions between different samples.…”

Section: Discussionmentioning

confidence: 99%

“…Because this enzyme is essential for the maintenance of cell function, it is generally assumed that it is constitutively expressed at similar levels in all cell types and tissues and many studies make use of its encoding gene as control without proper validation of its presumed stability of expression. Indeed, there have been numerous reports demonstrating that the mRNA levels of GAPDH gene are not constant, where the experimental treatment or condition influences its expression (Schmittgen and Zakrajsek 2000). The use of ribosomal RNA has been recommended as internal standards for mRNA quantification studies as the various RNA transcripts are generated by a distinct polymerase (Paule and White 2000) and their levels are less likely to vary under conditions that affect the expression of mRNAs (Barbu and Dautry 1989).…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis

Gonçalves

Cairney

Marôco

et al. 2005

Planta

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In order to determine the suitability of reference or housekeeping genes as internal controls in real-time reverse transcriptase PCR (RT-PCR) assays for quantification of target mRNAs, we studied the levels of expression of four candidate reference genes in maritime pine by real-time RT-PCR. The expression levels obtained for glyceraldehyde-3-phosphate-dehydrogenase, 18S ribosomal RNA, eukaryotic translation initiation factor eIF4AII and ubiquitin in nine stages of embryo development revealed that none of the genes tested proved to be suitable as an internal control. Copy number quantification of the four transcripts showed an average relative variation of seven fold. We propose that the combination of a precise method for RNA quantification, internal controls for monitoring RT reaction and PCR efficiency and a robust external standard curve can guarantee a reliable absolute quantification of mRNA transcripts in real time RT-PCR. This approach may avoid the controversy in the use of housekeeping genes and may assume special significance in tissues undergoing developmental changes.

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High‐Throughput Real‐Time Quantitative Reverse Transcription PCR

Bookout

Cummins

Mangelsdorf

et al. 2005

CP Molecular Biology

109

115

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This unit describes the use of real-time quantitative PCR (QPCR) for high-throughput analysis of RNA expression. The topics covered include: design and validation of QPCR primers and probes for both SYBR Green-and TaqMan-based assays (see Support Protocol); the standard curve method (see Basic Protocol 1); an efficiency-corrected Ct (cycle time, also called cycle threshold or crossing point) method (see Basic Protocol 2); and the comparative cycle time, or Ct method (see Alternate Protocol). While the unit describes the use of the Applied Biosystems 7900HT (high-throughput, 384-well) instrument, the protocols may be utilized for any real-time PCR instrument. The highthroughput design allows analysis of the levels of transcript from a number of genes of interest (GOIs) at one time by using the appropriate primer set for each gene. (Within this unit, the term GOI will refer to the actual gene of interest as well as its RNA product or cDNA copy.)Because of the simplicity of the mathematical application, the standard curve method (Basic Protocol 1) is the most basic and straightforward QPCR assay described in the unit. In this method, standard curves are constructed for all of the GOIs from which RNA expression is being measured, and linear regression analysis is applied to interpolate arbitrary unknown sample values. The standard curve assay may be performed even if the PCR amplification efficiencies of the primer sets (as determined by the template dilution assay in the Support Protocol) are not equal, since correction for unequal efficiencies is intrinsic to the linear regression formula. One drawback of the standard curve method is that standard curves must be run for each of the primer sets on an assay plate. This results in less space on the plate for the unknown samples, and requires the use of additional reagents. This use of resources is particularly excessive when the PCR amplification efficiencies of the primer sets have been determined to be 100% and relative fold-change is the preferred outcome of the measurements. In such cases, the Ct method (see Alternate Protocol) should be employed instead. Another limitation is that unless the levels of all of the GOIs in the cDNAs used to construct the standard curves are known, the relative concentration of one GOI cannot be compared to that of another GOI. If comparison between the levels of different GOIs (without the knowledge of the relative level of the transcripts in the standards) is desired, the efficiency-corrected Ct method (see Basic Protocol 2) should be applied.The efficiency-corrected Ct method builds upon the standard curve method by incorporating PCR efficiency (E) into the quantity calculations. The standard curve slopes are used to calculate PCR efficiency according to the relationship E = 10 (-1/slope) . The efficiency has a maximum value of 2 for perfect doubling of the PCR template (see Basic Protocol 2 for an in-depth explanation of E). Note that in this method the standard curve is used only to determine slope and not to interpolate the RNA...

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Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR

Cited by 1,090 publications

References 5 publications

UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines

UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines

Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis

High‐Throughput Real‐Time Quantitative Reverse Transcription PCR

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