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Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a.

Sikand K, Singh J, Ebron JS, Shukla GC - PLoS ONE (2012)

Bottom Line: The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked.Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data.Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio, United States of America.

ABSTRACT
Results of overexpression or downregulation of a microRNA (miRNA) on its target mRNA expression are often validated by reverse-transcription and quantitative PCR analysis using an appropriate housekeeping gene as an internal control. The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked. Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and β-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

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miR-644a downregulates GAPDH and β-actin protein expression.(A, B and C) Representative western blots showing the expression of GAPDH, β-actin and STAT2 in LNCaP, 293T and HeLa cells treated with indicated amounts of miR-644a mimic or negative control (NC) mimic for 48 hours. STAT2 expression was used as a loading control. (D, E and F) Quantitation of GAPDH protein expression in the respective lanes as shown in A, B and C. (G, H and I) Quantitation of β-actin protein expression in the respective lanes as shown in A, B and C. Three independent western blots were used for the quantification of protein expression. The signal intensities of bands were measured using ImageJ software. The GAPDH or β-actin expression in each lane was determined by normalizing GAPDH or β-actin band intensity to STAT2 band intensity. Data are plotted as mean ± SE of three independent experiments.
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pone-0047510-g002: miR-644a downregulates GAPDH and β-actin protein expression.(A, B and C) Representative western blots showing the expression of GAPDH, β-actin and STAT2 in LNCaP, 293T and HeLa cells treated with indicated amounts of miR-644a mimic or negative control (NC) mimic for 48 hours. STAT2 expression was used as a loading control. (D, E and F) Quantitation of GAPDH protein expression in the respective lanes as shown in A, B and C. (G, H and I) Quantitation of β-actin protein expression in the respective lanes as shown in A, B and C. Three independent western blots were used for the quantification of protein expression. The signal intensities of bands were measured using ImageJ software. The GAPDH or β-actin expression in each lane was determined by normalizing GAPDH or β-actin band intensity to STAT2 band intensity. Data are plotted as mean ± SE of three independent experiments.

Mentions: While studying the effect of a panel of miRNAs on target mRNA expression in prostate cancer cell lines, we found that the use of GAPDH or β-actin as normalization control in case of miR-644a treatment yielded misleading results causing us to suspect the repression of GAPDH and β-actin expression by miR-644a. To confirm this, we evaluated the effect of miR-644a on GAPDH and β-actin expression in a panel of three cell lines: LNCaP (human prostate cancer), HEK 293T (human embryonic kidney) and HeLa (human cervical cancer). Cells were transfected with miR-644a mimic or negative control (NC) mimic. GAPDH and β-actin mRNA/protein levels were measured 48 hours post-transfection. 18S ribosomal RNA (rRNA) expression was used as an internal control for normalization of GAPDH and β-actin mRNA expression. GAPDH and β-actin protein levels were normalized to signal transducer and activator of transcription 2 (STAT2) expression. Our computational analysis confirmed that STAT2 open reading frame including 5′ and 3′ UTRs does not appear to contain any miR-644a target site that follows established miRNA-target mRNA base-pairing rules. Nevertheless, we investigated if STAT2 mRNA expression is affected by miR-644a transfection. As seen in figure 1A, miR-644a reduced GAPDH mRNA levels by 50% to 90% as compared to NC mimic in LNCaP, 293T and HeLa cells. Similar reduction was observed in β-actin mRNA expression in the three cell lines transfected with miR-644a mimic (Figure 1B). As expected, miR-644a failed to inhibit the expression of STAT2 mRNA (Figure 1C). These data confirmed that GAPDH and β-actin are targets of miR-644a and STAT2 is not. Furthermore, western blots showed the repression of GAPDH and β-actin protein levels by miR-644a transfection in LNCaP, 293T and HeLa cells (Figure 2). Taken together, these data provide evidence for miR-644a-mediated regulation of GAPDH and β-actin expression and hence, indicate the unsuitability of these housekeeping genes as internal controls in experiments involving miR-644a. A recent study [4] designed to evaluate the androgen receptor-targeting miRNAs has also noted the repressive effect of miR-644a on GAPDH mRNA expression; however, this study did not report any repressive effect of miR-644a on β-actin mRNA expression, which was used as an endogenous control instead of GAPDH. Also, no further attempts were made to evaluate if GAPDH is a direct target of miR-644a.


Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a.

Sikand K, Singh J, Ebron JS, Shukla GC - PLoS ONE (2012)

miR-644a downregulates GAPDH and β-actin protein expression.(A, B and C) Representative western blots showing the expression of GAPDH, β-actin and STAT2 in LNCaP, 293T and HeLa cells treated with indicated amounts of miR-644a mimic or negative control (NC) mimic for 48 hours. STAT2 expression was used as a loading control. (D, E and F) Quantitation of GAPDH protein expression in the respective lanes as shown in A, B and C. (G, H and I) Quantitation of β-actin protein expression in the respective lanes as shown in A, B and C. Three independent western blots were used for the quantification of protein expression. The signal intensities of bands were measured using ImageJ software. The GAPDH or β-actin expression in each lane was determined by normalizing GAPDH or β-actin band intensity to STAT2 band intensity. Data are plotted as mean ± SE of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472982&req=5

pone-0047510-g002: miR-644a downregulates GAPDH and β-actin protein expression.(A, B and C) Representative western blots showing the expression of GAPDH, β-actin and STAT2 in LNCaP, 293T and HeLa cells treated with indicated amounts of miR-644a mimic or negative control (NC) mimic for 48 hours. STAT2 expression was used as a loading control. (D, E and F) Quantitation of GAPDH protein expression in the respective lanes as shown in A, B and C. (G, H and I) Quantitation of β-actin protein expression in the respective lanes as shown in A, B and C. Three independent western blots were used for the quantification of protein expression. The signal intensities of bands were measured using ImageJ software. The GAPDH or β-actin expression in each lane was determined by normalizing GAPDH or β-actin band intensity to STAT2 band intensity. Data are plotted as mean ± SE of three independent experiments.
Mentions: While studying the effect of a panel of miRNAs on target mRNA expression in prostate cancer cell lines, we found that the use of GAPDH or β-actin as normalization control in case of miR-644a treatment yielded misleading results causing us to suspect the repression of GAPDH and β-actin expression by miR-644a. To confirm this, we evaluated the effect of miR-644a on GAPDH and β-actin expression in a panel of three cell lines: LNCaP (human prostate cancer), HEK 293T (human embryonic kidney) and HeLa (human cervical cancer). Cells were transfected with miR-644a mimic or negative control (NC) mimic. GAPDH and β-actin mRNA/protein levels were measured 48 hours post-transfection. 18S ribosomal RNA (rRNA) expression was used as an internal control for normalization of GAPDH and β-actin mRNA expression. GAPDH and β-actin protein levels were normalized to signal transducer and activator of transcription 2 (STAT2) expression. Our computational analysis confirmed that STAT2 open reading frame including 5′ and 3′ UTRs does not appear to contain any miR-644a target site that follows established miRNA-target mRNA base-pairing rules. Nevertheless, we investigated if STAT2 mRNA expression is affected by miR-644a transfection. As seen in figure 1A, miR-644a reduced GAPDH mRNA levels by 50% to 90% as compared to NC mimic in LNCaP, 293T and HeLa cells. Similar reduction was observed in β-actin mRNA expression in the three cell lines transfected with miR-644a mimic (Figure 1B). As expected, miR-644a failed to inhibit the expression of STAT2 mRNA (Figure 1C). These data confirmed that GAPDH and β-actin are targets of miR-644a and STAT2 is not. Furthermore, western blots showed the repression of GAPDH and β-actin protein levels by miR-644a transfection in LNCaP, 293T and HeLa cells (Figure 2). Taken together, these data provide evidence for miR-644a-mediated regulation of GAPDH and β-actin expression and hence, indicate the unsuitability of these housekeeping genes as internal controls in experiments involving miR-644a. A recent study [4] designed to evaluate the androgen receptor-targeting miRNAs has also noted the repressive effect of miR-644a on GAPDH mRNA expression; however, this study did not report any repressive effect of miR-644a on β-actin mRNA expression, which was used as an endogenous control instead of GAPDH. Also, no further attempts were made to evaluate if GAPDH is a direct target of miR-644a.

Bottom Line: The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked.Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data.Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio, United States of America.

ABSTRACT
Results of overexpression or downregulation of a microRNA (miRNA) on its target mRNA expression are often validated by reverse-transcription and quantitative PCR analysis using an appropriate housekeeping gene as an internal control. The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked. Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and β-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

Show MeSH