1

Gene expression analysis in superficial bladder cancer: comparison of two suitable

endogenous reference genes

Running title: Gene expression normalization in superficial bladder cancer

Authors: Paula A. Videira1, Dário Ligeiro

2, Manuela Correia

1, Hélder Trindade

1,

Affiliation of the authors:

1 Departamento de Imunologia FCM-UNL, Campo Mártires da Pátria 130, 1169-056

Lisboa, Portugal.

2 Centro de Histocompatibilidade do Sul, Lisboa, Portugal.

To whom correspondence should be addressed:

Paula A. Videira, Departamento de Imunologia FCM-UNL, Campo Mártires da Pátria

130, 1169-056 Lisboa, Portugal.

Telephone: +351 218 803 045; Fax: +351 218853480

Email: pvideira.imuno@fcm.unl.pt

2

Keywords

Superficial bladder cancer; normalization; endogenous control gene; real-time reverse

transcription PCR; β-actin; GAPDH

Abstract

Gene expression studies demand that data is normalized to an endogenous control gene.

The selection of this control is not straightforward, in particular for tumour tissue

samples. We have measured the expression of genes engaged in the immune response,

in matched samples of superficial bladder cancer relative to normal urothelium. The β-

actin and GAPDH genes were selected from a panel of common reference genes as the

most stably expressed, and were, hence, used for normalization. Even so, we found that

in some samples, the immunity genes expression, when normalized to either β-actin or

to GAPDH was opposing. The combination of both reference genes was clearly more

adequate to be considered. Nevertheless, the gene expression studies should be regarded

as qualitative rather than quantitative. Our study stresses the required prudence when

selecting endogenous control genes. The gold standard normalization is most probably

obtained with the mean expression of multiple endogenous controls.

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Introduction

The fluorescence-based real-time reverse transcription PCR (RT-PCR) is a widely used

technology for the quantification of mRNA level, and is becoming a critical tool for

basic and clinical research [1]. RT-PCR is now applied to identify genes which correlate

with disease and the inflammatory response to disease. Its advances in cancer have

allowed the identification of tumour markers and have improved the tumour diagnosis

and prognosis [2].

For the RT-PCR, it is mandatory to relate, i.e., to normalize each gene expression to a

stably expressed reference gene, referred as endogenous control gene. The ideal

endogenous control is usually an housekeeping gene, which means its expression should

be sufficiently abundant, constant across samples and be unaffected by experimental

treatments [1, 3]. Including an invariant endogenous control gene in the assay, corrects

for sample to sample variations in RT-PCR efficiency and sample amount differences;

which is essential for the reliability of any relative RT-PCR experiment [4].

Nevertheless, the selection of the appropriate endogenous control is not straightforward.

The genes described as having an invariant expression by the great majority of the

studies, have also been shown to be cell or tissue dependent [4] and to vary in certain

circumstances, such as cell proliferation [5], hypoxia or experimental treatment [6] and

neoplastic cell growth [7, 8].

Therefore, when it comes to the identification of genes with altered expression in

tumours, the use of endogenous control genes for normalization may have increased

pitfalls.

In the present study, we have analysed the expression of ten genes in samples of

superficial bladder cancer relative to normal urothelium. The study was initiated in

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December 2005 and aimed, originally, to study the expression of genes engaged in the

immune response in superficial bladder cancer. The samples were obtained from thirty

patients. For normalization, we have analysed a panel of eleven common endogenous

control genes, and the GAPDH and β-actin genes were selected for showing the most

constant and stable expression in these tissues. Even though, the data here presented

shows the unanticipated variations in relative gene expression found in some samples,

when using these endogenous control genes alone or combined.

Materials and methods

Patient tissue samples

Matched pairs of cancerous bladder tissue and normal urothelium samples were

obtained from 30 patients with a mean age of 69,3 years (range 45 to 88) with

diagnosed superficial bladder cancer (tumour staged at pTa and pT1, according to the

International Union Against Cancer - UICC) after transurethral resection of the tumours.

Tissue samples were immediately immersed in RNAlater® RNA Stabilization Reagent,

stored at 4º C, overnight and then preserved until further processing at -20º C. Informed

consent was obtained from all patients and the use of the tissue material was approved

by the local ethics committee.

RNA Isolation and Characterization

Preserved tissue samples (20 to 50 mg wet weight) were potter homogenized and the

total RNA was isolated using the GenElute Mammalian Total RNA Purification kit and

DNAase treatment (Sigma), according to the manufacturer’s instructions.

RNA concentrations and A260-to-A280 ratios were measured in a spectrophotometer and

only samples with A260/A280 ratios between 1,9 and 2,1 were considered further.

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Between 250 to 500 ng of total RNA were reverse transcribed with random primers,

using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA).

Expression of endogenous control genes

The expression of endogenous control genes was assessed using the TaqMan Human

Endogenous Control Plate (Applied Biosystems) according to manufacturer’s protocol.

This plate evaluates the expression of eleven common endogenous control genes [18S

rRNA (18S), Acidic ribosomal protein (ARP), Beta-actin (β-actin), Cyclophilin (CYC),

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Phosphoglycerokinase (PGK),

β2-Microglobulin (B2m), β-Glucoronidase (GUS), Hypoxanthine ribosyl transferase

(HPRT), Transcription factor IID TATA binding protein (TBP) and Transferrin receptor

(TfR)]. Matched samples of cancerous and normal urothelium of three randomly chosen

patients were used for testing. In these assays, exactly 500 ng of total RNA was reverse

transcribed into cDNA as described.

Gene Expression Measurements

RT-PCR was performed in a 7500 Fast system (Applied Biosystems) using TaqManFast

Universal PCR Master Mix, primers and Taqman probes provided by Applied

Biosystems and following the manufacturer’s instructions. The analyzed genes were

MIP-1α (Hs00234142_m1), MCP-1 (Hs00234140_m1), MCP-2 (Hs00271615_m1), IP-

10 (Hs00171042_m1), MIG (Hs00171065_m1), CD1A (Hs00233332_m1), CD1B

(Hs00233507_m1), CD1C (Hs00233509_m1), CD1D (Hs00174321_m1) and CD1E

(Hs00229421_m1). Each reaction was performed in duplicate. All genes, including the

endogenous controls, and the matched samples of cancerous and noncancerous tissues

were always analyzed in the same run to exclude between-run variations. The number of

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PCR cycles needed to reach the fluorescence threshold in each sample was defined as

the cycle threshold (Ct).

The relative expression for each gene was calculated by using the formula 2-∆∆Ct

,

according to the method described by Livak and Schmittgen [9]. ∆Ct stands for the

difference between the Ct of the amplification curve of the target gene and that of the

endogenous control gene. The efficiency of the amplification reaction for each primer-

probe was above 95% (as determined by the manufacturer).

Data analysis

Data was analysed for statistical significance using the Student’s t-test and ANOVA One-

way (GraphPad Prism Version 4.0). Correlations between variables were tested using the

Spearman correlation analysis. P values < 0,05 were considered as significant.

To calculate the expression stability of the candidate endogenous control genes, the freely

available software packages geNorm™, version 3.5 [[10],

http://medgen.ugent.be/~jvdesomp/genorm/], NormFinder18 [[11],

http://www.mdl.dk/publicationsnormfinder.htm] and BestKeeper1 [[12], http://www.gene-

quantification.de/bestkeeper.html] were used.

Results

Selection of reference genes

In order to select two of the most appropriated endogenous control gene to initiate the

study of the relative gene expression in superficial bladder cancer, we have first

performed a MEDLINE search for the most common genes used by others to normalize

the gene expression in bladder cancer or other neoplasms. According to our search, the

β-actin or the GAPDH genes were the most frequently used for normalization in the

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majority of neoplasms, including in bladder cancer measurements [13-15]. As in recent

publications, these genes are presented as inappropriate for general bladder cancer

(tumours staged between pTa and pT4) [11, 16], we have analysed few patient samples,

for the expression of eleven genes, commonly used as reference genes. In order to rank

the reference genes, based on their expression stability, determining the most stable

genes for normalization, we have used three different methods, the geNorm,

NormFinder and BestKeeper software. geNorm calculates the gene expression stability

measure, M, for a reference gene as the average pairwise variation for that gene with all

other tested reference genes. Among the analysed reference genes, the most stable were

ranked as follows, according to the lowest M value (Table 1): GAPDH > β-actin > TfR>

HPRT> TBP> PGK> CYC> ARP> GUS> B2m> 18S. All these genes reach a

reasonable high expression stability with M values less than 1, below the default limit of

M = 1,5 [10].

NormFinder is another tool which estimates the intra- and intergroup variation and

combines both results in a stability value for each reference gene [11]. Genes with the

lowest stability value are the most stable within the group studied. The results of the

NormFinder analysis applied to our data have given a ranking identical to the geNorm

(Table 1).

According to these results, the 18S is the most unsuited reference gene and GAPDH is

the most stable gene, closely followed by the β-actin.

BestKeeper uses a pair-wise correlation analysis of all pairs of candidate genes [12]. In

a first analysis of the data, based on the inspection of raw Ct values, BestKeeper

calculates variations of all reference gene (SD [± Ct]) and a low SD is expected for

useful reference genes. In a second analysis, BestKeeper estimates the correlations in

the expression levels between all the possible candidates. As this algorithm determines

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the best suited standards, out of only ten candidates, we have excluded the 18S gene due

to its worst ranking with the previous tools. The BestKeeper analysis has also revealed

some similarity with the geNorm and NormFinder analysis. In fact, GAPDH has the

lowest SD [± Ct] value and a good correlation coefficient with the remaining analysed

genes (Table 1). Although B2m and GUS genes present SD [± Ct] values lower than the

β-actin gene, those genes have a very low correlation coefficient β-actin is, therefore,

the second best suited reference gene, according to the BestKeeper analysis.

In accordance with to the obtained results, we have chosen GAPDH and β-actin as the

best endogenous control genes to normalize our gene expression study.

Expression levels of target genes normalized with GAPDH and/ or β-actin genes

The expression of ten genes related with the immune response in bladder cancer was

analysed in 30 patients. The study was encompassed by genes coding for the CD1A,

CD1B, CD1C, CD1D and CD1E molecules, which are important molecules for

presenting lipid antigens [17]; and also genes coding for chemokines, MCP-1 and MCP-

2, also known as monocyte chemoattractant proteins [18], MIP-1α, the macrophage

inflammatory protein-1-alpha [19], MIG, the monokine induced by gamma-Interferon

and IP-10, the Interferon-inducible protein-10 [20].

The gene expression profiles in cancerous bladder tissue relative to normal urothelium

were compared using either the β-actin or the GAPDH gene for normalization and the

results revealed a significantly different variance (p < 0,001) for all the analysed genes

(Fig. 1). When using the geometric mean of the expression of β-actin and GAPDH to

normalize the data, the overall profile was also significantly different (p < 0,05) from

the expression profiles obtained using β-actin or GAPDH genes individually, in all

genes except for the MCP-1 and MCP-2 genes (Fig. 1).

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Still, it is interesting to note that, whereas the cytokine genes present a divergent profile,

all CD1 molecules are downregulated in a great majority of the cancerous bladder tissue

relative to normal urothelium (Fig. 1). These results are probably related with fact that

tumourigenesis usually induces the downregulation of antigen presenting molecules in

the tumour tissue. This downregulation is known to have an important contribution to

the tumour escape to the immune surveillance [21].

Expression variation of the GAPDH and/ or β-actin reference genes

Given the differences found for the expression of target genes, when normalized to β-

actin, GAPDH or both, we have examined the expression variances of these reference

genes in the 30 samples. When comparing matched pair samples, we have observed that

both genes amplified in a distinct way. In urothelium, both genes have an interval of

amplification curve Ct, i.e., the differences between β-actin and GAPDH Ct (Ctβ-actin -

CtGAPDH) with a median of -0,08, ranging from -3,02 to 1,86 (Fig. 2A). On the other

hand, in cancerous bladder tissue, the Ctβ-actin - CtGAPDH have a median of 1,01, ranging

from -1,85 to 4,96 (Fig. 2A). Comparing these differences in matched pairs of

cancerous and normal patient’s samples, the Ctβ-actin - CtGAPDH in cancerous tissue

differed with a median of 1,02 Cts, ranging from -1,98 to 4,72, from the Ctβ-actin -

CtGAPDH of normal urothelium (Fig. 2B). In fact, 12 out of the 30 samples presented Ctβ-

actin - CtGAPDH differences between cancerous and normal urothelium higher than one Ct

(Fig. 2B). Among them, the samples 2, 4, 5, 7, 11, 20, 28 and 30, presented Ctβ-actin -

CtGAPDH differences higher than two Cts (Fig. 2B). Consequently, these samples gave

rise to more suspicious results regarding the relative expression of the analyzed genes,

obtained after normalizing to β-actin, GAPDH or both (Fig. 1). As an example, in

patient 2, the analysis of the CD1A expression, normalized to β-actin gave a two fold

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increased expression in tumour relative to urothelium. Yet, if the data is normalized

with GAPDH, the expression drops to 0,08, which mean 12,5 fold less expression in

tumour relative to urothelium (Fig. 1). When considering the geometric mean, the

obtained results are 0,38, which mean 2,6 less expression in tumour relative to

urothelium.

Disregarding the values and in view of the qualitative expression; in other words,

considering only, whether the expression is increased or decreased in cancer relative to

urothelium, the obtained results are more consensual. As an example, in patient 20, the

data of the expression of CD1B, CD1C, CD1D, CD1E, MIP-1α, MCP-1 and MCP-2

genes was always decreased in cancer relative to urothelium, independently of the

choice of the endogenous control (Fig. 2).

Discussion

The use of suitable endogenous control gene for cell or tissue gene expression

measurements is still a matter of debate. The prevalent opinion is to previously test

several control genes and select the most constant and the gold standard normalization

is now considered to measure the expression of several endogenous control genes and

normalize using their mean expression [10].

For the study here presented, we have selected the β-actin and GAPDH genes, based

essentially on the results obtained from a test using a panel of endogenous controls.

Even so, these genes present significant expression variability in the analysed samples

and in, some measurements, the obtained results were contradictory. Our observations

have showed two aspects. 1) Caution should always be taken even when using

endogenous control genes selected as suitable reference genes from a panel of common

housekeeping genes. 2) When using one or two combined controls, the relative

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expression should be regarded as qualitative rather than quantitative. 3) The most

reliable normalization is obtained with the mean expression of more than one

endogenous control. Even though, the use of only two control genes may be insufficient

and the use of multiple housekeeping genes should lead to more reliable results.

The selection of suitable endogenous control genes are costly and time consuming

procedures. Therefore, many researchers easily assume, as endogenous control gene, a

gene already referred by others.

As the β-actin and GAPDH have been the most frequently used genes for normalizing

expression measurements, many authors have focused on the study of their reliability as

reference genes. As a result, there are nowadays an increasing number of publications

reporting the pitfalls of using them as endogenous control [16, 22]. Additionally other

genes have been proposed as more accurate housekeeping genes for analysing cancer

samples. As an example, in general bladder cancer, two important works report the most

suitable reference genes,[10, 11]. Since these works are based on distinct panels of

reference genes, each indicate their own most suitable reference gene. So far, the

reference genes reported by these authors were not analysed by others in bladder cancer.

It is likely that a more profound research reveals that those genes, described as

invariable in some studies, are found to be regulated in other studies, even with the

same type of analysed tissue. It is reasonable to consider that there is no gold standard

for analysing the expression in cancer. Therefore the use of multiple endogenous genes

is surely the most accurate approach for gene expression measurements in cancer.

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Legends to figures

Figure 1. Relative quantification (RQ) of the expression of genes associated with the

immune response in 30 patients with superficial bladder cancer. RQ values correspond

to the expression of a defined gene in cancerous tissues relative to normal urothelium.

Samples were normalized to the geometric mean of β-actin and GAPDH expression

(black columns), to the β-actin (grey columns) or to the GAPDH gene (white columns).

Values above or below 1 indicate, respectively, overexpression or downregulation of a

specific gene in the tumour tissue relative to the healthy tissue.

Figure 2. Differential expression of the β-actin and GAPDH in matched samples of

normal urothelium and bladder cancer tissue. A: Differences between β-actin and

GAPDH Ct (Ctβ-actin - CtGAPDH) either in urothelium or in tumour tissue. B: Differences

between the Ctβ-actin - CtGAPDH of tumour tissue and the Ctβ-actin - CtGAPDH of the matched

urothelium of each patient (B).

Figure 1

CD1A

0,00

0,01

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

RQ

CD1C

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RQ

CD1D

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CD1E

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IP-10

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Figure 1 (continue)

MCP-1

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Figure 2

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Urothelium Tumour

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A B

Matched patient samples

1

Table 1. Candidate endogenous control genes for normalization and their expression

stability values calculated by geNorm, NormFinder and BestKeeper softwares.

Gene geNorm* NormFinder BestKeeper**

18S 0,735 0,488

B2m 0,441 0,252 0,68

0,180

GUS 0,399 0,211 0,72

0,230

CYC 0,383 0,176 1,53

0,975

PGK 0,353 0,150 0,96

0,826

GAPDH 0,306 0,073 0,54

0,808

HPRT 0,334 0,131 0,88

0,896

β-actin 0,307 0,092 0,73

0,810

ARP 0,390 0,191 1,10

0,947

TBP 0,337 0,139 0,89

0,751

TfR 0,328 0,103 1,02

0,834

A low stability value corresponds to a high expression stability of the respective gene, therefore to the

best endogenous control genes for normalization. * In GeNorm, the stability value is designated as M

value. ** Measures of the SD [± Ct], standard deviation of the Ct and correlation coefficients (bellow

values) obtained with the Bestkeeper analysis.