Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors
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Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Here, we evaluated the most suitable housekeeping gene for RNA expression analysis in CDCs cultured under normoxia, hypoxia or with prolyl-4-hydroxylase inhibitors (PHDIs), from both neonatal and adult rats, to determine the effects of ageing and different culture conditions on the stability of the housekeeping gene for CDCs. Six candidate housekeeping genes, [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp)] were evaluated in this study. Analysis using geNorm and NormFinder revealed that GAPDH was the most constant housekeeping gene among all genes tested under normoxia for both neonatal and adult CDCs, whereas Actb was the most stable housekeeping gene under hypoxia. For the PHDI-treated CDCs, overall, GADPH, Actb and β2M were more consistently expressed, whereas HPRT-1, RPLP-1 and Tbp showed unstable expression. The ranking for β2M, HPRT-1 and RPLP-1 stability was different for neonatal and adult cells, indicating that expression of these genes was age-dependent. Lastly, independent of age or culture conditions, Tbp was the least stable housekeeping gene. In conclusion, a combination of Actb and GADPH gave the most reliable normalization for comparative analyses of gene transcription in neonatal and adult rat CDCs preconditioned by hypoxia or PHDIs.
KeywordsHousekeeping genes Cardiosphere-derived cells Hypoxia Prolyl-4-hydroxylase inhibitors
Stem cells were first identified within the heart in 2003 , challenging the generally accepted paradigm that the heart is a post mitotic organ [2, 3]. In 2004, Messina et al.  developed a method to isolate stem cells from human and murine heart, expanding them as cardiospheres (CSp). Such CSp were clonogenic, expressed stem and endothelial progenitor cell antigens/markers, and appeared to have the properties of adult cardiac stem cells. The CSp were expanded to obtain a reasonable number of cardiosphere-derived cells (CDC) for transplantation . CSp and CDCs derived from heart explant culture in vitro caused myocardial regeneration and functional improvement when injected into the infarcted mouse heart in vivo [4, 5].
Preconditioning of stem cells to enable survival in the hypoxic environment has been postulated to improve cell production efficiency and potency for myocardial repair . The implantation of hypoxic (5% O2) CDCs into infarcted hearts of mice in vivo resulted in greater cell engraftment and better functional recovery than with conventionally cultured (normoxic) CDCs . Hypoxia triggers a number of physiological and cellular adaptations to reduced oxygen, with many processes involved in oxygen homeostasis being mediated by the hypoxia-inducible factor (HIF) transcriptional complex, which is negatively regulated by the prolyl-4-hydroxylase (PHD) enzyme. The PHD enzyme is a conserved subfamily of dioxygenases that uses oxygen and 2-oxoglutarate as a co-substrate and iron as a co-factor to catalyse the post-translational hydroxylation of specific prolyl residues of HIF-α subunits [7, 8, 9]. Upon hydroxylation, the HIF-1α subunit is recognized by the von Hippel–Lindau protein which targets the subunit to proteasomal degradation . The sensitivity of PHD hydroxylase capacity depends on its co-substrate and co-factor. Thus, limited oxygen supply or inhibition of the 2-oxoglutarate or iron could inhibit PHD activity and thus potently activate the HIF response [10, 11, 12, 13].
Here, we cultured CDCs under hypoxia (2% oxygen) or used three different prolyl-4-hydroxylase inhibitors (PHDIs) for CDC HIF stabilization. The PHDIs used in this study included dimethyloxaloylglycine (DMOG) – a cell permeable, competitive inhibitor of 2-oxoglutarate , ethyl 2-(2,3-dihydroxybenzamido) acetate (EDBA) – an aspirin metabolite that activates the HIF system via generic 2-oxoglutarate oxygenase inhibition , and 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid (BIC) – a specific PHD inhibitor patented by FibroGen Inc. (FG2216), which has been used in clinical trials as a pro-angiogenic compound acting via the HIF-1α system .
List of reference genes and target gene used in the present study
Forward primer 5′ to 3′
Reverse primer 5′ to 3′
Gene accession number
Function of the protein coded by this gene
Oxidoreductase in glycolysis and gluconeogenesis
Cytoskeletal structure protein
Hypoxanthine phosphoribosyl-transferase 1
Recycle purines in cells
Highly conserved proteins that are involved in cell motility, structure and integrity
60S acidic ribosomal protein large P1
TATA box binding protein
Transcription factor that binds to TATA box located at upstream of the transcription start site in eukaryotic gene promoters
Hypoxia induced factor-1 alpha
Transcription factors that respond to reduced oxygen changes in the cellular environment
RNA and cDNA quality
CDC sample groups
Age of animals
(a) Neonatal (1–3 days)
(i) Normoxia (21% O2) (control)
(b) Adult (4 months)
(ii) Hypoxia (2% O2)
(iii) Normoxia + 1 mM DMOG for 24 h
(iv) Normoxia + 0.5 mM EDBA for 24 h
(v) Normoxia + 30 μM BIC for 24 h
List of reference genes and target gene used in the present study
Correlation coefficient (R2)
The expression profiles of candidate housekeeping genes
HKGs stability test using geNorm and Normfinder
To identify the housekeeping gene with the most stable expression in the CDC samples, two software-based methods were used: geNorm and NormFinder.
geNorm is a well published Microsoft Excel based program designed to validate stability of housekeeping gene expression. The underlying principles and calculations are described in Vandesompele et al. . In brief, geNorm ranks gene stability by an average expression stability value (M), which is the average pairwise variation of a single candidate housekeeping gene with all other candidate housekeeping genes. More stable gene expression is indicated by lower M values.
However, a limitation of this software is that the M value measured is the expression of a single gene as a ratio of the other genes tested. Thus, we could not resolve the best gene out of the two best performing genes, GADPH and Actb. In addition, geNorm analysis relies on the principle that the two best performing genes are identical in all samples, regardless of the experimental conditions or treatments, which is not necessarily true. To address this problem, NormFinder software was also used.
Ranking of candidate HKGs and their expression stability, V in the different sample groups of the study evaluated using NormFinder
DMOG 1 mM
EDBA 0.5 mM
BIC 30 μM
(a) Neonatal CDCs
(b) Adult CDCs
(c) Combined (neonatal and adult CDCs)
HIF-1α mRNA expression normalized by different HKGs
This study clearly illustrates the differences in stability of housekeeping genes in rat CDCs cultured under normoxia (21% O2), hypoxia (2% O2) or with the PHDIs: DMOG, EDBA or BIC.
Preconditioning of cells under hypoxia or in the presence of an iron chelator (EDBA), a 2-oxoglutarate analogue (DMOG) or a specific prolyl hydroxylation inhibitor (BIC), inhibits PHD enzyme activity and subsequently activates the HIF transcriptional complex [10, 11, 12, 13]. Analysis of protein levels in hypoxic and PHDI-treated CDCs revealed a significant increase in HIF-α expression, compared to normoxic CDCs, in both the neonatal and adult group, indicating that the hypoxic challenge to the cells was successful. Analysis of mRNA levels in hypoxic and PHDI-treated CDCs using qRT-PCR is, however, not as straightforward as the protein analysis. The analysis of qRT-PCR data required normalization using a consistently expressed housekeeping gene.
RPLP-1, which plays a role in the elongation step of protein synthesis , was the most abundantly expressed gene in the rat CDCs, whereas HPRT-1 (an enzyme which recycles purine in cells)  and Tbp (a transcription factor which binds to the TATA-box DNA sequence) , were the least expressed genes. Genes with extensive or low expression are not suitable reference genes for qRT-PCR due to their extreme transcription expression. Our conclusions were validated by the geNorm and Normfinder analysis, which showed that these three genes were relatively unstable, compared to GAPDH, β2M and Actb, which showed moderate expression in rat CDC.
To evaluate the effect of ageing on HKG stability, we grouped the samples into neonatal and adult groups and analyzed the data either separately or as a combined set using geNorm. In both approaches, we found that a combination of GAPDH and Actb were best to be used as housekeeping gene, while the Tbp was the least stable among all the candidate genes. The instability of β2M, HPRT-1 and RPLP-1 at different stages of heart development is a drawback in their use as housekeeping genes.
Normalization with multiple reference genes is becoming more prevalent, but studies that apply this normalization approach are limited. Based on geNorm analysis, we found that an optimum of two housekeeping genes was needed to perform a valid normalization. To confirm this, we evaluated the expression of HIF-1α mRNA in the study samples normalized to only one housekeeping gene or to a combination of the two best housekeeping genes as selected by geNorm analysis. Interestingly, only the data set normalized to the geometric mean of GADPH and Actb genes showed a significant increase in HIF-1α expression in every sample. Thus, two housekeeping genes seemed optimal for accurate normalization in this study.
In our experiments, the sample sets included different stages of heart development (neonatal versus adult rat hearts), different oxygen tensions (normoxic versus hypoxic) and three different PHDI drug treatments (DMOG, EDBA or BIC). For NormFinder analysis, we grouped the samples within each experiment, taking into account the genes associated with cardiac progenitor cells in normoxic and hypoxic CDCs or between physiologically-induced hypoxia using hypoxic culture and pharmaceutically-induced hypoxia using PHDIs. We found that GADPH was the most stable gene expressed in the normoxic cell group. However, the expression of this gene was unstable in hypoxic CDCs where Actb was the most stable gene expressed. For the PHDI-treated samples, there were different best housekeeping genes selected for each inhibitor, yet overall, GADPH, Actb and β2M were the most stable reference genes. Although different housekeeping genes were selected for the different experimental conditions, when the stability of housekeeping genes were considered over all groups, the best housekeeping genes revealed by NormFinder analysis was GADPH and Actb combined.
In conclusion, both geNorm and NormFinder analysis revealed that GAPDH and Actb were the best housekeeping genes for this study. geNorm also indicated that two housekeeping genes were required for valid qRT-PCR data normalization. In most cases, using multiple housekeeping genes resulted in more accurate and reliable normalization compared with the use of only one reference gene. Thus, we suggest that Actb and GADPH genes should be both used as the housekeeping genes in qRT-PCR involving normoxia, hypoxic and PHDI-treated neonatal and adult rat CDCs.
Sprague–Dawley (SD) rats were used for all experiments. All experiments were performed with approval from The University of Oxford Animal Ethics Review Committees and the Home Office (London, UK).
Primary cardiac stem cell culture
CDCs were isolated and expanded according to a protocol described previously . In brief, neonatal (1–3 days) and adult (4 months) SD rat hearts were isolated, and minced into small explants in 0.05% trypsin–EDTA (Invitrogen). Explants were plated on fibronectin-coated petri dishes with 1.5 ml of complete explant medium (CEM, Additional file 3) to generate explant-derived cells (EDCs). EDC, once confluent, were isolated and seeded onto poly-d-lysine coated 24-well plates with cardiosphere growth medium (CGM, Additional file 3) to form spherical cell clusters, known as Csp. Loosely attached Csp were harvested and plated onto fibronectin-coated T75 flasks with CEM, at a density of 1 × 104 cells/flask for expansion into cardiosphere derived cells (CDCs). All the cells were cultured in incubator under 21% O2 and 5% CO2, at 37°C, unless otherwise stated.
CDC preconditioned with hypoxia and PHDIs
For hypoxic-preconditioned cells, CDCs were cultured under 2% O2 (hypoxia) and 5% CO2 at 37°C using an incubator (Wolf Laboratories, UK) adjustable to different oxygen concentrations by infusion of nitrogen (N2). The O2 concentration was monitored continuously using an O2 sensor. For PHDI-preconditioning, normoxic CDCs (cultured under 21% O2 and 5% CO2, at 37°C) were treated with 1 mM of DMOG, 0.5 mM of EDBA or 30 μM of BIC for 24 h at 80% cell confluency.
Western blot analysis for HIF-1α protein
Normoxic, hypoxic and PHDI-preconditioned CDCs were lysed in cell lysis buffer containing protease inhibitor. The homogenate was boiled and centrifuged for 5 min, the supernatant frozen and stored at −80°C. The lysate (50 μg/μl) was subjected to SDS–polyacrylamide gel (10%) electrophoresis, and protein was subsequently transferred to a nitrocellulose membrane (Pall Life Science, Mexico). The membrane was incubated overnight with rat HIF-1α primary antibody diluted (1:2000) in 5% milk in TBS-Tween (Novus, NB100–479). The secondary antibody used was goat anti-rabbit conjugated to horseradish peroxidase (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA). The immunoreactive protein signal was visualized by an ECL plus detection kit (Perkin Elmer). Protein bands were quantified using Un-Scan-It, Version 6.1 (Silk Scientific, USA).
Primer design and optimization
Primers were designed using Primer3 software based on interpretation of GenBank or Ensembl Genome Browser search results (Table 2). Primer specificity was enhanced by designing a primer pair that flanked the exon–exon border of the gene of interest, to avoid the primers amplifying genomic DNA that may be contained in samples. Primer specificity was confirmed by blasting the primer sequence against genomic databases available at NCBI. No significant homology sequence should be found, to ensure gene specific amplification rather than amplification of several transcripts with one primer pair.
RNA extraction and DNase treatment
Total RNA was extracted from cultured CDCs using Trizol reagent (Sigma) according to the manufacturer’s instructions. DNase treatment was performed using Turbo DNA-free (Ambion) to degrade any DNA present. The concentration and purity of RNA was determined by measuring the absorbance at 260 nm (A260) and 280 nm (A280) using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies Inc., USA). A ratio of A260/A280 ≈ 2.0 is generally accepted as pure RNA.
cDNA was synthesized from the RNA template using AB high capacity transcriptase kit (Applied Biosystem). Every 1 μg RNA sample was reverse transcribed using 1 μl reverse transcriptase, 2 μl random primer, 0.8 μl dNTPs (10 mM each), 2 μl buffer and topped up by RNase free water to a total volume of 20 μl. The reaction mixture was subjected to incubation for 10 min at 25°C, 120 min at 37°C and 5 s at 85°C.
Quantitative RT-PCR (qRT-PCR)
qRT-PCR amplification was performed using Applied Biosystems StepOnePlus Real-Time PCR System (AB International, CA). The qRT-PCR mastermix was prepared by adding 10 μl AB Sybrgreen PCR mastermix (AB International, CA), 1 μl reverse primer, 1 μl forward primer, 1 μl cDNA and 7 μl distilled water. The total volume for each real-time PCR reaction was 20 μl. The PCR program was set up with an initial heat activation step at 95°C for 10 min. Then, 40 cycles of thermocycling were performed with a denaturation step at 95°C for 15 s, an annealing step at 60°C for 30 s and an extension step at 72°C for 30 s. Fluorescence was measured at the end of each extension step. After amplification, a melting curve was acquired by heating the product at 4°C/s to 95°C, cooling it at 4°C/s to 70°C, keeping it at 70°C for 20 s, and then slowly heating it at 4°C/s to 95°C. Fluorescence was measured through the slow heating phase. Melting curves were used to determine the specificity of PCR products.
Analysis of housekeeping gene expression stability
Raw Ct values of housekeeping genes were converted to linear values compatible with the geNorm and NormFinder programs. For comparison of housekeeping gene stability between groups, the software geNorm, version 3.4 (Visual Basic application tool for Microsoft Excel)  was used, while for housekeeping genes stability comparison within group, NormFinder (a Microsoft Excel Add-in)  was used. Both programs were used according to developer’s recommendations as published.
Analysis of target gene expression
Raw Ct values of the target gene (HIF-1α) were analyzed using two different methods to determine the importance of normalization to different housekeeping gene(s) in qRT-PCR data analysis.
Data analysis using 2−ΔΔCt method
Simple relative quantification of target gene expression normalized to one housekeeping gene was performed using the 2−ΔΔCt method. Derivation of the equation, including assumptions, experimental design and validation test, have been described in Applied Biosystem User Bulletin No. 2 (P/N 4303859). Analyses of gene expression data using this method have appeared in the literature [38, 39, 40].
Data analysis using geNorm method
Relative quantification of target gene expression normalized to more than one housekeeping gene was performed using the geNorm method. In this method, target gene expression was normalized to the geometric mean expression of the best combination of housekeeping genes subjected to expression stability analysis using geNorm software. Analyses of gene expression data using this method have appeared in the literature [23, 41, 42, 43].
The software program, SPSS version 18 was used for further statistical analysis of gene and protein expression among experimental groups. In all cases expression data were compared by one-way analysis of variance (ANOVA) (statistical significance < 0.05). The significance level (α) was set at 0.05.
This work was supported by the British Heart Foundation (grant number PG/07/059/23259). SCT and KKY thank the Malaysian Ministry of Higher Education for studentships.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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