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, 7 (10), e47510

Housekeeping Gene Selection Advisory: glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) and β-Actin Are Targets of miR-644a

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Housekeeping Gene Selection Advisory: glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) and β-Actin Are Targets of miR-644a

Kavleen Sikand et al. PLoS One.

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. miR-644a downregulates GAPDH and β-actin mRNA expression.
(A and B) Quantitative real-time PCR analysis of GAPDH and β-actin mRNA expression in LNCaP, 293T and HeLa cells transfected with miR-644a mimic or negative control (NC) mimic. (C) In order to demonstrate that the repression of GAPDH and β-actin mRNA expression is a consequence of specific targeting by miR-644a, the effect of miR-644a was checked on a computationally predicted non-target gene, STAT2. STAT2 mRNA expression was determined by quantitative real-time PCR analysis in LNCaP, 293T and HeLa cells transfected with miR-644a mimic or NC mimic. GAPDH, β-actin and STAT2 mRNA expression was normalized to 18S rRNA expression. Data are plotted as mean ± SE of three independent experiments.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. Conservation of miR-644a target site.
Panels A and B show alignments of GAPDH and β-actin 3′ UTR sequences containing miR-644a binding site in 7 mammalian species. miR-644a target site sequence is shown in gray box and seed binding region is shown in bold. Stars indicate conserved nucleotides in the target sequence in at least 5 out of 7 species.
Figure 4
Figure 4. GAPDH is a direct target of miR-644a.
(A) Schematic representation of firefly luciferase reporter construct containing GAPDH 3′ UTR with either wild type (WT) or mutant (MUT) miR-644a target site. The miR-644a target site in GAPDH 3′ UTR is italicized and underlined. In the MUT-3′ UTR construct, 5 nucleotides (1183–1187) in the seed binding region of the target site were mutated to their complementary nucleotides (shown in bold) in order to disrupt miR-644a binding. (B) Luciferase reporter assay in CHO-K1 cells cotransfected with WT-3′ UTR or MUT-3′ UTR construct and miR-644a mimic (2 nM) or negative control (NC) mimic (2 nM) as indicated. Renilla luciferase reporter plasmid was cotransfected in all cases as a control for transfection efficiency. Luciferase activity is plotted as a ratio of firefly to renilla luciferase activity. Each bar represents mean ± SE of three independent experiments.
Figure 5
Figure 5. β-actin is a direct target of miR-644a.
(A) Schematic representation of firefly luciferase reporter construct containing β-actin 3′ UTR with either wild type (WT) or mutant (MUT) miR-644a target site. The italicized and underlined sequence in β-actin 3′ UTR represents the miR-644a target site. In the MUT-3′ UTR construct, 2 nucleotides (1562–1563) in the seed binding region of the target site were mutated to their complementary nucleotides (shown in bold) in order to disrupt miR-644a binding. (B) Luciferase reporter assay in CHO-K1 cells cotransfected with WT-3′ UTR or MUT-3′ UTR construct and miR-644a mimic (2 nM) or negative control (NC) mimic (2 nM) as indicated. Renilla luciferase reporter plasmid was cotransfected in all cases as a control for transfection efficiency. Luciferase activity is plotted as a ratio of firefly to renilla luciferase activity. Each bar represents mean ± SE of three independent experiments.

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Associated data

Grant support

Research in GCS laboratory is supported by grants from National Science Foundation (NSF) and DoD Prostate Cancer Research Program. JS was supported by NSF grant 0842606 and JSE was supported by DoD-USAMRAA grant W81XWH-11-1-0204. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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