Approach for defining endogenous reference genes in gene expression experiments

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 329 (2004) 293–299 www.elsevier.com/locate/yabio

Approach for deWning endogenous reference genes in gene expression experiments J.J. García-Vallejo,a B. Van het Hof,a J. Robben,a J.A.E. Van Wijk,b I. Van Die,a D.H. Joziasse, and W. Van Dijka,¤ a

Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Centre, 1081 BT, Amsterdam, The Netherlands b Department of Pediatrics, Vrije Universiteit Medical Centre, 1081 BT, Amsterdam, The Netherlands Received 22 December 2003 Available online 6 May 2004

Abstract The quantiWcation of gene expression by real-time polymerase chain reaction (PCR) has revolutionized the Weld of gene expression analysis. Due to its sensitivity and Xexibility it is becoming the method of choice for many investigators. However, good normalization protocols still have to be implemented to facilitate data exchange and comparison. We have designed primers for 10 unrelated genes and developed a simple protocol to detect genes with stable expression that are suitable for use as endogenous reference genes for further use in the normalization of gene expression data obtained by real-time PCR. Using this protocol, we were able to identify human proteosome subunit Y as a reliable endogenous reference gene for human umbilical vein endothelial cells treated for up to 18 h with TNF
, IL-4, or IFN and for B cells isolated from healthy controls and patients suVering from IgA nephropathy. Other optional endogenous reference genes that can be considered are phosphomannomutase (PPMM) and actin for endothelial cells and glyceraldehyde-3-phosphate dehydrogenase and PPMM for B cells.  2004 Elsevier Inc. All rights reserved. Keywords: Endogenous reference gene; Housekeeping gene; Gene expression analysis; Real-time PCR; HUVEC; Endothelial cells; B cells; IgA nephropathy

The quantiWcation of gene expression, either at the mRNA or at the protein level, is one of the most extended research techniques in the life sciences. Several methods have been used in past years to provide high sensitivity and speciWcity in the quantiWcation of gene expression. To date, there is an array of possibilities with diVering characteristics and applications that include Northern blotting, RNase protection assay, in situ hybridization, serial analysis of gene expression, realtime PCR, and cDNA microarray technology. Quantitative real-time PCR is the most sensitive and Xexible method of the aforementioned methods [1,2] for the quantiWcation of gene expression and presents many advantages over the other techniques, such as less handson time, technical ease, low reagents cost, and high throughput. ¤

Corresponding author. Fax +31-20-444-8144. E-mail address: [email protected] (W. Van Dijk).

0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.02.037

The needs for normalization (expression of the data in the same units) and calibration are inherent to the problem of quantiWcation in any biochemical setting. In the case of real-time PCR, normalization can be achieved with the use of endogenous (already existing in the sample) or exogenous (characterized RNA or DNA spiked into the sample during its preparation) controls, whereas calibration relies on reference samples. The most extended endogenous control is the use of maintenance genes or housekeeping genes1. These genes are classically deWned as constitutively expressed genes that

1 Although housekeeping genes can be used as endogenous reference genes in gene expression experiments, in our opinion the term “housekeeping gene” should not be used as a synonym for “endogenous reference gene” since it does not directly imply that a certain gene is not subjected to any kind of regulation in the situation of interest. Hence, the term endogenous reference gene will be used throughout this article.

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are necessary to maintain the homeostasis of cells and thus are normally expressed at a constant level. By taking advantage of this property it is then possible to compare the relative expression of a target gene to that of an endogenous reference gene (ERG)2 in two or more diVerent samples. An alternative to the use of endogenous reference genes is to use rRNAs as endogenous reference. However, rRNAs can be used only when total RNA is isolated and the cDNA synthesis is primed with random hexamers. In some studies plasmids containing the amplicon of the target or endogenous reference gene have been used to generate a standard curve and express the data as a relative quantiWcation of absolute data [3,4]. Once normalization has been performed, calibration introduces additional information in the sense that it allows investigators to directly compare how a treatment (or diVerent organs, tissues, disease conditions, etc) inXuences the expression of a certain gene with respect to a control. It can be easily achieved by establishing a normalized measurement as a denominator for further comparisons (e.g., treated vs nontreated, organ A/B/C vs organ D, disease vs normal). Although the principle for normalization by using an endogenous reference seems straightforward it does not lack complications [1,5,6] and some discrepancies remain unsolved [7–10]. The discrepancies are based on the fact that some endogenous reference genes do not vary in some studies, while they are clearly regulated in others. Thus, variables such as culture conditions, cell cycle, or treatment may have repercussions on the expression levels of an endogenous reference gene or rRNA [11]. To develop a method for deWning the most suitable endogenous reference gene for quantitative gene expression studies we designed a set of primers for 10 potential endogenous reference genes and applied a statistical approach to cluster the genes according to the variation of their expression levels. These genes were chosen from a database of stably expressed genes on the basis of their unlinked biological function or lack of coregulation. This novel method is independent of cDNA input and permits a clear distinction of nonregulated clusters of stably expressed genes. As a proof of principle, we applied this method to primary human umbilical vein endothelial cells (HUVEC) subjected to diVerent stimuli and to B lymphocytes isolated from healthy and IgA nephropathy patients.

Materials and methods Endothelial cells HUVEC were isolated from Wve healthy donors by a modiWcation of the method of JaVe et al. [12]. After delivery, umbilical cords were conserved in cord buVer (4 mM KCl, 140 mM NaCl, 11 mM Hepes, 12 mM D-glucose, and 100 U/mL penicillin–streptomycin; Biowhittaker, USA) for up to 6 days at 4 °C. BrieXy, cords were canulated in both ends and washed Wrst with warm cord buVer and second with warm M199 (Biowhittaker) supplemented with 100 U/mL penicillin–streptomycin. Then, 10 mL of a Wltered sterilized solution containing 135 U/ mL of collagenase type 2 (Worthington Biochemical, USA) in M199 was introduced into the cord and the canules were clamped. After 20 min of incubation in cord buVer at 37 °C, the solution was collected and centrifuged at 1500 rpm for 5 min. The cells were resuspended in M199 supplemented with 100 U/mL penicilin– streptomycin, 10% human serum, 10% newborn calf serum, 5 U/mL heparine (Leo Pharmaceutical Products, The Netherlands), and 150 g/mL bFGF (Sigma, The Netherlands) and plated in gelatin-coated plates. The cells were cultured to conXuency in the mentioned medium in a 5% CO2 atmosphere at 37 °C. When conXuency was reached, cells were trypsinized (0.18% trypsin, 10 mM EDTA) and plated again to 1/3rd of their density. In the second passage, cells were exposed to 100 U/ mL of TNF
, 100 U/mL of IL-1, 200 U/mL of IFN, 50 U/mL of IL-4 (Strahtmann Biotech, GmbH, Germany), or combinations of the cytokines for 2, 6, or 18 h. B cells Blood samples from 11 healthy controls and 11 patients with biopsy-proven IgA nephropathies were 1:1 diluted in PBS, and peripheral blood mononuclear cells were separated by density gradient centrifugation on Ficoll 400 (Amersham Biosciences Europe, GmbH, The Netherlands). The peripheral blood mononuclear cells were washed once in PBS/0.2% BSA and resuspended in 1 mL PBS/0.2% BSA. B cells were isolated using CD19conjugated magnetic beads (MACS, Miltenyi Biotec, Germany) according to the manufacturers instructions. The purity of the cell population was 195% by FACScan analysis using Xuorescein isothiocyanate-labeled CD19. Isolation of RNA and cDNA synthesis

2

Abbreviations used: ERG, endogenous reference gene; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buVered saline; BSA, bovine serum albumin; ERG, endogenous reference gene; Nt, normalized amount of target; PPMM, phosphomannomutase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HumPSY, human proteosome subunit Y.

mRNA was speciWcally isolated by capturing of poly(A+) RNA in streptavidin-coated tubes with a mRNA Capture kit (Boehringer Mannheim, GmbH, Germany) and cDNA was synthesized with the Reverse Transcription System kit (Promega, USA) following manufacturer’s guidelines. Cells (approximately 2 £ 105/

J.J. García-Vallejo et al. / Analytical Biochemistry 329 (2004) 293–299

well) were washed twice with ice-cold PBS and harvested with 200 L of lysis buVer. Lysates were incubated with biotin-labeled oligo(dT)20 for 5 min at 37 °C and then 50 L of the mix was transferred to streptavidin-coated tubes and incubated for 5 min at 37 °C. After washing three times with 250L of washing buVer, 30 L of the reverse transcription mix (5 mM MgCl2, 1£ reverse transcription buVer, 1 mM dNTP, 0.4 U recombinant RNasin ribonuclease inhibitor, 0.4 U AMV reverse transcriptase, 0.5 g random hexamers in 30 L nucleasefree water) was added to the tubes and incubated for 10 min at room temperature followed by 45 min at 42 °C. To inactivate AMV reverse transcriptase and separate mRNA from the streptavidin–biotin complex, samples were heated at 99 °C for 5 min, transferred to microcentrifuge tubes, and incubated in ice for 5 min. Then the samples were diluted 1:2 in nuclease-free water and stored at ¡20 °C until analysis. Real-time PCR Primers have been designed by using the computer software Primer Express 2.0 (Applied Biosystems, USA). The primer design strategy has been to situate primers within the coding sequence, exon-spanning when possible, with an optimal length of 20 nucleotides, a Tm between 58 and 60 °C, an amplicon length between 50 and 150 bp, and a limited number of G and C in the 3⬘ end of the oligonucleotides. The panel of genes chosen, with their GenBank accession numbers, is listed in Table 1. The PCRs were performed with the SYBR green method in a ABI 7900HT sequence detection system (Applied Biosystems, USA). The reactions were set on a 96-well plate by mixing 10 L of the 2£ concentrated

295

SYBR Green Master Mix (Applied Biosystems) with 5 L, of a primer solution containing 5 nmol/L of both primers and 5 L of a cDNA solution corresponding to 1/60th of the cDNA synthesis product. The thermal proWle for all reactions was 2 min at 50 °C, followed by 10 min at 95 °C and then 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The Xuorescence monitoring ocurred at the end of each cycle. The primer speciWcity was computer tested by homology search with the human genome. Additionally, dissociation curve analysis was performed in every run. For this purpose, the temperature of the block was increased from 60 to 95 °C at a rate of 1.75 °C/min while continuously monitoring Xuorescence. The plot of the Wrst derivative of the decrease in Xuorescence with respect to temperature showed in all cases one single peak at the Tm predicted by the Primer Express software (Applied Biosystems). The eYciency [13] of the primers was tested in preliminary experiments with a pool of cDNA of the samples used in the present study and maintained an average of 90% (range: 80.3–96.6). Data analysis The Ct value is deWned as the number of PCR cycles where the Xuorescence signal exceeds the detection threshold value, which is Wxed as 10 times the standard deviation of the Xuorescence during the Wrst 15 cycles and typically corresponds to 0.2 relative Xuorescence units. This threshold is set constant throughout the study and corresponds to the log linear range of the ampliWcation curve. The diVerence between the Ct of the target gene and the Ct of the ERG, Ct D CtTarget ¡ CtERG, is used to obtain the normalized

Table 1 List of primers used in the present study Gene

GeneBank

Elf-1


J04617

MLN51

X80199

Ubch5B

U39317

 Actin

X00351

CYES

M15990

HPRT

M31642

HumPSY

D29012

L32

X03342

GAPDH

M33197

PPMM

U86070

Primers ⬘

Fwd:5 -TCGGGCAAGTCCACCACT Rev:5⬘-CCAAGACCCAGGCATACTTGA Fwd:5⬘-ACCTCCAGTCCCAGAAACCA Rev:5⬘-TCCAATTCTGTTCTGCTATATTTAGTTGT Fwd:5⬘-GTACTCTTGTCCATCTGTTCTCTGTTG Rev:5⬘-GTCCATTCCCGAGCTATTCTGTT Fwd:5⬘-GCTCCTCCTGAGCGCAAG Rev:5⬘-CATCTGCTGGAAGGTGGACA Fwd:5⬘-CAGGTATGGTGAACCGTGAAGTAC Rev:5⬘-TCAATTCATGGAGGGATTCTGG Fwd:5⬘-TCGAGCAAGACGTTCAGTCCTG Rev:5⬘-TCGAGCAAGACGTTCAGTCCTG Fwd:5⬘-ACCTGATGGCGGGAATCAT Rev:5⬘-ATCATACCCCCCATAGGCACT Fwd:5⬘-CAACATTGGTTATGGAAGCAACA Rev:5⬘-TGACGTTGTGGACCAGGAACT Fwd:5⬘-CCATGTTCGTCATGGGTGTG Rev:5⬘-GGTGCTAAGCAGTTGGTGGTG Fwd:5⬘-AAGCGTGGAACCTTCATCGA Rev:5⬘-TCCCGGATCTTCTCTTTCTTGTC

Function

Ref

Protein synthesis

[17]

Oncogenesis (?)

[18]

Ubiquitinilation

[19]

Cytoskeleton

[20]

Tyrosine kinase

[21]

Nucleotide metabolism

[22]

MHC class I

[23]

Ribosomal protein

[24]

Glycolysis

[1]

Sugar-donnor metabolism

[25]

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amount of target (Nt), which corresponds to 2¡Ct [14]. The Nt reXects the relative amount of target transcripts with respect to the expression of the ERG. The statistical parameters employed have been mean, median, standard deviation (SD), and coeYcient of variation of the mean (CV), calculated as CV D 100 SD/mean, which allows the comparison of the variability in the measurements.

Results The quantiWcation of the expression of the 10 selected genes was assayed by SYBR-green-based real-time PCR in all the samples. In the case of group A, human umbilical vein endothelial cells were subjected to diVerent

Scheme 1. Protocol for the selection of an endogenous reference gene (ERG). cDNA is synthesized from RNA isolated from samples obtained under every condition of interest for the study in which the ERG is going to be used. Subsequently, the samples are assayed for 10 unrelated ERGs. The Wrst of the genes is set as potential ERG and the Nt of all the other genes is calculated. On this set of data, the CV is calculated. The process is repeated for all of the genes. Finally a matrix of CVs that deWnes high- and low-variability gene clusters is obtained (as in Fig. 2). The Wnal ERG is chosen based on the median of the CV of the diVerent potential ERGs.

stimuli (TNF
, IL-4, IFN) for either 2, 6, or 18 h. Group B corresponded either to healthy individuals or to IgA nephropathy patients. The analysis of the raw data (Ct values) was performed in 10 cycles (Scheme 1). In each cycle, one of the selected genes was set as the potential endogenous reference gene and the Nt was calculated for all the others. Subsequently, the CV of the Nt was obtained for each gene, and then the median of all the CVs was determined. Once all the genes were considered “potential” endogenous reference genes, the table obtained (Table 2) allowed ranking of the genes according to the median of the CVs. Medians lower than 10 deWne low-variability genes while medians over 15 deWne high-variability genes. From all the genes with low-variability, a Wnal endogenous reference gene can be chosen according to the lowest variation (lowest median of the CVs). Additionally other parameters such as the expression level of the gene of interest (Fig. 1) can be considered. A summary of the raw data (Ct values) is shown in Fig. 1, where the median, quartiles, and extremes of the Ct are displayed. Also the approximate expression levels can be substracted. The normality of the sample distribution (Kolmogorov–SmirnoV) could not be assumed in any group; consequently nonparametric statistics were applied. No statistical diVerences (Kruskal–Wallis) were observed among the diVerently treated samples in group A (N D 29) nor in the patient and control samples in group B (N D 22) (Mann–Whithey). Both in group A and in group B (Table 2), only three genes cluster as highly variable, Elf-1, MLN51, and cYES. From these clusters, the lowest median of the CV showed that the best endogenous reference gene was HumPSY in both groups. However, a more detailed examination of Table 2 reveals that in group A the seven genes clustered as low-variability have the median of the CVs between 5.4 and 7.6, so that probably any of the diVerent genes in that cluster would be a very reliable endogenous reference gene. In the group B, although HumPSY has the lowest median, L32 has more CVs below 5. With this approach we were able to identify HumPSY as a reliable endogenous reference gene for HUVECs treated for up to 18 h with TNF
, IL-4, or IFN and for B cells from healthy controls and patients suVering from IgA nephropathy. Other optional endogenous reference genes that can be considered are PPMM and actin in HUVECs and GAPDH and PPMM in B cells. Fig. 2 is shown to illustrate the importance of the lack of variation of a gene to be considered an endogenous reference. The same Ct values for E-Selectin were normalized by GAPDH, HumPSY, and MLN51. If we would have chosen MLN51 as the endogenous reference, we would have overestimated the up-regulation with TNF
and we would have missed the modulation by IFN of the TNF
-induced up-regulation of E-Selectin, which has been suYciently demonstrated before [15].

J.J. García-Vallejo et al. / Analytical Biochemistry 329 (2004) 293–299

297

Table 2 CoeYcients of variation (CV) of the normalized amount of target (Nt) for every potential endogenous reference gene as obtained in HUVECs and B cells Potential ERGs

Actin

Group A (HUVECs) Actin Elf-1 25.2 GAPDH 5.8 HPRT 3.0 HumPSY 4.1 L32 6.7 MLN51 41.4 PPMM 5.3 UbcH5B 7.6 CYES 24.5 Group B (B cells) Actin Elf-1 18.58 GAPDH 6.88 HPRT 7.43 HumPSY 6.28 L32 6.87 MLN51 27.84 PPMM 6.38 UbcH5B 7.22 CYES 20.35

Elf-1

GAPDH

HPRT

HumPSY

L32

MLN51

PPMM

UbcH5B

cYES

Median

Clustering

33.2

5.2 23.6

3.1 26.8 5.8

4.1 25.5 4.6 3.8

6.8 26.3 7.7 7.5 5.4

23.4 26.5 23.0 23.2 21.7 20.5

5.1 26.3 5.5 4.6 2.2 6.3 39.7

7.3 26.4 9.3 7.9 6.8 6.8 38.5 7.3

18.6 30.7 17.2 18.3 18.5 21.2 38.8 19.0 21.0

6.8 26.3 7.6 7.5 5.4 6.8 39.7 6.0 7.6 25.0

LV HV LV LV LV LV HV LV LV HV

13.02 23.18 11.25 11.91 12.34 13.07 26.42 13.73 12.76

11.94 20.04 6.88 8.20 5.84 12.21 25.38 7.61 10.89 21.33

33.1 33.0 32.8 33.7 38.4 33.0 33.7 32.4 35.41 35.31 35.17 35.02 35.84 40.32 35.23 35.86 34.60

5.0 4.9 8.5 39.7 6.0 9.5 20.8 10.10 17.24 4.47 3.94 7.85 24.23 6.35 7.15 15.88

3.5 6.4 39.9 4.7 7.1 26.8 11.32 20.04 4.50 4.79 5.34 24.65 7.56 4.84 21.39

5.6 39.0 2.3 6.3 24.2 13.66 18.97 4.37 8.20 13.52 23.98 6.09 11.43 18.40

43.1 5.9 6.4 24.9 9.04 20.38 6.28 5.44 5.25 27.27 7.61 4.08 21.33

21.5 21.2 30.0 21.19 21.83 21.27 20.54 20.05 18.70 19.68 19.04 29.74

7.4 25.0 11.94 19.79 6.09 9.26 4.74 12.21 25.38 10.89 19.51

25.5 9.77 20.78 6.97 5.40 5.84 3.96 23.42 8.25 21.53

HV LV LV LV HV LV HV

The data represent all conditions in HUVECs (group A) and B cells from patients and controls (group B) as deWned under Materials and methods. In every sample, the Nt of the set of ERG tested (labeled horizontally) was calculated using each of them as a potential ERG (labeled vertically) at a time; subsequently, the CV of all the Nt for every potential ERG was determined (see Scheme 1 for more details on the method). The median of all the CVs for every potential endogenous reference gene is included as a help to deWne clusters of high (HV) and low (LV) variability. See Materials and methods for deWnition of CV and Nt.

Fig. 1. Summary plot of the data. The box represents the interquartile range which contains 50% of the values. The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers (open triangles). A line across the box indicates the median.

Discussion Normalization is as important in gene expression studies as the techniques used to obtain the data. It allows researchers to compare expression proWles obtained in diVerent laboratories or under diVerent conditions. Although is clear that the use of an endogenous reference, often referred to as internal control genes, is the gold standard, there is little agreement on the conditions that a gene has to meet to be considered

an “endogenous reference gene.” Nevertheless, it is clear that it should be highly expressed and it should not vary among diVerent conditions or cell types. In this report, we describe for the Wrst time a method to identify one or more endogenous reference genthat can be applied in the normalization of gene expression studies. The present panel of es unrelated endogenous reference genes was chosen based on previous studies. Some of the genes have been used profusely in many studies [1];

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The alternatives to the use of endogenous reference genes, namely, the use of copies/cell or copies/g of RNA, still represent problems which are diYcult to overcome. One of them is the eYciency of the two necessary steps prior to the real-time PCR quantiWcation, the RNA isolation and the cDNA synthesis, which can be aVected by many factors. On the other hand, total RNA can be aVected by the Xuctuations of its main constituent, rRNA, and RNA quantiWcation can be largely inXuenced by proteins, free nucleotides, and other possible contaminants in the preparation. The inclusion of this protocol in any gene expression experiment will improve reliability of results and will allow researchers to directly compare their own results with published real-time PCR data.

References Fig. 2. Human umbilical vein endothelial cells were stimulated with TNF
(100 IU/mL) or TNF
(100 IU/ml) and IFN (200 IU/mL). After 6 h, cells were harvested, RNA was isolated, and cDNA was synthesized. The expression levels of E-Selectin were assayed considering GAPDH, HumPSY, and MLN51 as possible endogenous reference genes.

such is the case of the genes for the glycolysis enzyme glyceraldehyde-3-phosphate dehydrogenase, for the cytoeskeleton protein  actin, or for the nucleotide metabolism enzyme hypoxanthine-guanine phosphoribosyltransferase [5]. Seven more genes known to be functionally independent were chosen from a list of 535 endogenous reference genes identiWed by microarray in 11 human tissues [16] (Table 1). Most importantly, all genes belong to completely unrelated cell functions, as it has been impossible to Wnd studies linking them; so it is extremely unlikely to exhibit regulated covariation. Since it is expected that 10 constitutively expressed genes, functionally independent and necessary for cell life, should not be regulated in the same manner, it is then possible to isolate clusters of nonregulated endogenous reference genes by excluding those whose expression is clearly modiWed in diVerent circumstances. The diVerences in the Ct of nonregulated endogenous reference genes are likely to depend on diVerences in the input of cDNA or in the starting amount of material rather than on the speciWc treatment or eVect that we are studying. The advantage of this method relies on the fact that it is based mainly on the biological signiWcance of gene expression, while it also considers statistical indicators. Furthermore, it is independent of the cDNA content, so it is not aVected by possible errors in the RNA quantiWcation or the cDNA synthesis. Additionally, only a little amount of cDNA is needed for the protocol proposed (Scheme 1), since these are highly expressed genes, so this should not be an obstacle for large expression proWling experiments.

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