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Reliable Housekeeping Gene Combination for Quantitative PCR of Lymph Nodes in Patients with Prostate Cancer

IGOR TSAUR1, MARKUS RENNINGER2, JOERG HENNENLOTTER2, ELSIE OPPERMANN3, MARITA MUNZ2, URSULA KUEHS2, ARNULF STENZL2 and DAVID SCHILLING1,2

Departments of 1Urology and 3Surgery, University Hospital Frankfurt, Frankfurt, Germany; 2Department of Urology, University Hospital Tuebingen, Tuebingen, Germany

Abstract. Background: To reliably compare the results of gene and profiling. Major advantages of qRT-PCR are the low probe expression studies, the expression of the target gene should be volume, cost-effectiveness, reproducibility of results, objective normalized to the expression of a reference gene. For lymph node evaluation and the possibility to quantitatively investigate gene metastases of prostate cancer, no data on polymerase chain expression (3). Although qRT-PCR is highly sensitive and reaction (PCR) normalization have yet been reported. We aimed precise, the accuracy may be hampered by variations in assay to determine the most reliable reference gene combination for design, inconsistent data analysis and normalization (4). To this purpose in patients with prostate cancer. Materials and minimize misleading result interpretation and provide Methods: Ten histologically- positive and ten negative lymph comparability between different tissue samples, the expression nodes of patients with prostate cancer were analyzed of the target gene is usually normalized to the expression of a respectively. Expression of six candidate reference genes was reference gene analyzed simultaneously in the same comparatively assessed with quantitative Real-time PCR. The experimental setting. These ”housekeeping” genes are most stably-expressed gene combination was determined with putatively expressed uniformly at steady levels, independently geNorm software version 3.4. Results: Hypoxanthine of internal and external conditions. However, expression levels phosphoribosyltransferase-1 (HPRT1) and TATA box binding of housekeeping genes may also be subject to regulation protein (TPB) were found to be the most stably expressed genes, depending on the tissue type, cell cycle and differentiation state with their combination having an expression stability value of of the target cells (5, 6). M=0.17. Conclusion: Gene combination HPRT1 and TPB has For LN metastases of PC, no data on the quality of the potential to be utilized for normalization in gene profiling normalization have been reported so far. For this reason, we assessment of metastatic and non-metastatic pelvic lymph node sought to determine the most reliable reference gene tissue from patients with prostate cancer. combination for comparative analysis of gene expression in LN specimens from patients with PC in a pilot study. Based Emerging evidence indicates that distinct alterations in gene on previous investigations by Ohl et al. (7) and a current expression profile are critical for tumor cell dissemination and MEDLINE research, we selected six frequently used reference formation of metastases during tumorigenesis of prostate genes namely aminolevulinate, delta-, synthase-1 (ALAS1), cancer (PC) (1, 2). Metastasis to locoregional lymph nodes tubulin, alpha 1b (K-ALPHA-1), glyceraldehyde-3-phosphate (LN) represents one of the first clinically measurable events in dehydrogenase (GAPDH), TATA box binding protein (TBP), systemic disease progression. To more thoroughly understand hypoxanthine phosphoribosyltransferase-1 (HPRT1) and beta- the key steps in tumor biology, assessment of molecular 2-microglobulin (B2M), for quantitative mRNA analysis in LN characteristics of tumor in different compartments, such as LN, specimens with and without the presence of PC metastases. is indispensable. Quantitative real-time polymerase chain The publicly available analytical software program geNorm reaction (qRT-PCR) is often applied for gene expression studies (http://medgen.ugent.be/~jvdesomp/genorm/) was used to statistically evaluate the suitability of the different reference genes.

Correspondence to: David Schilling, MD, Department of Urology, Materials and Methods Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. E-mail: [email protected] Patients and tissue samples. After receiving the approval of the study protocol by the Ethics Committee of the University of Key Words: Lymph node metastases, molecular staging, polymerase Tuebingen (no. 151/2006V), tissue samples of 10 lymph nodes with chain reaction, prostate cancer, reference gene. and 10 without histologically proven metastases were examined.

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Lymph nodes were derived from patients undergoing radical annealing and extension respectively. The amplification and detection prostatectomy including pelvic lymph node dissection for biopsy- of K-ALPHA-1 was performed with TaqMan Gene Expression Master proven PC between 06/2006 and 08/2007 at the Department of Mix according to the manufacturer’s instructions (Applied Urology of the University Hospital of Tuebingen. For this purpose, Biosystems). The final reaction volume of 10 μl consisted of 2× lymph node specimens were dissected immediately after removal Master Mix (5.0 μl), Primer Mix (2 pmol/μl respectively; 1.0 μl), and 2×2 mm tissue samples were snap-frozen in liquid nitrogen and probe (2 pmol/μl; 1.0 μl), water (1.0 μl) and prediluted cDNA (2.0 μl). stored at –80˚C until further processing. Before RNA extraction, a The cycle conditions were as follows: a step of the initial denaturation microdissected slice of each tissue sample was subjected to for 10 s at 95˚C followed by 40 cycles of 15 s at 95˚C for template immunocytochemistry for pancytokeratin to verify the presence of denaturation and 60 s at 60˚C for primer annealing and extension metastasis. Tumor stage was determined according to the seventh respectively. All experiments were performed with StepOnePlus Real edition of the TNM classification (8). Tumor grading was performed Time System (Applied Biosystems). The PCR efficiency was detected according to the Gleason score (9). for each gene by 1:10 RNA dilution of a known PC sample in four steps. In order to minimize variability of measurements, malignant and RNA isolation. For total RNA extraction, tissue samples stored nonmalignant samples were investigated as paired samples in one PCR at –80˚C were bruised under continuous nitrogen cooling and run to exclude inter-run variations. homogenized by a TissueRupter (Qiagen, Hilden, Germany). RNA All RNA samples were assayed for their concentration, purity isolation was performed by RNeasy Lipid Tissue Kit (Qiagen) and integrity. To check the size of PCR products and exclude the according to the manufacturer’s protocol. The concentration of the formation of primer dimers and non-specific DNA synthesis, PCR isolated RNA was measured by NanoDrop ND-1000 products were transferred onto a 2% agarose gel containing tris- spectrophotometer (Peqlab, Erlangen, Germany). An A260 reading borate-EDTA (TBE) buffer and ethidium bromide. The samples of 1.0 is equivalent to an RNA concentration of about 40 μg/ml were diluted with 6× DNA loading dye (Fischer Scientific, RNA. RNA concentration was adjusted to 200 ng/μl. The samples Schwerte, Germany) and were isolated by electrophoresis at 65 mA; were aliquoted and stored at –80˚C. a 100 bp DNA ladder (Fischer Scientific) was used to size PCR products. DNA bands were visualized at 320 nm. First-strand cDNA synthesis. The cDNA synthesis was performed according to the manufacturer’s protocol by Transcriptor First Strand Statistical analysis. Statistical analyses were performed with JMP cDNA Synthesis Kit (Roche, Penzberg, Germany) using 1.5 μl of total (SAS, Cary, NC, USA). Statistical significance was investigated by RNA (300 ng) together with 2.0 μl 5× buffer, 1.0 μl dNTPs (10 mM paired and unpaired Student’s t-tests. As a common practice of using respectively), 1.0 μl random-primer, 0.25 μl RNase Inhibitor, 0.25 μl only one housekeeping gene for normalization of RT-PCR is often reverse transcriptase and 4.0 μl water per reaction sample. The reaction accompanied by significant errors leading to unreliable data mixture was then incubated at 25˚C for 10 min (primer annealing) and interpretation, combination of reference genes was assessed. For at 55˚C for 30 min (reverse transcription of RNA to cDNA). Finally, comparison of the stability of candidate reference genes, the the reverse transcriptase was deactivated by heating the reaction software geNorm, version 3.4 (10), a widely applied algorithm and mixture for 5 min at 85˚C. The mixture was diluted with 25 μl RNase a Visual Basic application tool for Microsoft Excel (11), was used. free water and immediately used as PCR template. The software was developed by Vandesompele and co-workers (10) and is based on a comparative analysis of the reference gene Design of oligonucleotide primer. The measurements were expression in a set of candidate genes. For each candidate gene, performed with commercially available primers and TaqMan probes log2-transformed expression ratios are calculated pairwise to all for ALAS1, GAPDH, TBP, HPRT1 and B2M. The primer and probe other remaining genes for the given tissue on the basis of non- for K-ALPHA-1 were newly designed. The TaqMan probes were 5’- normalized qRT-PCR results. After determination of the pairwise labeled with the reporter fluorescent dye 6-carboxy-fluorescein variation as a parameter for the standard deviation of the ratio in a (FAM) and had the quencher dye 6-carboxy-tetramethyl-rhodamine panel of tissue samples, average pairwise variation with other (TAMRA). In order to achieve the most efficient qRT-PCR reference genes is determined as a gene-stability measure (M) for amplification, criteria for the correct primer choice were melting each gene. The lowest M values, reflective of the lowest standard temperature, optimal length (18-22 nucleotides), content of deviation and variation, indicate the best gene expression stability. guanosine/cytosine (40-60%) and a relatively short amplicon (60- The software excludes the most instably expressed gene and 150 base pairs). To avoid non-specific primer annealing, the primer calculates new M values, finally resulting in a combination of two sequences were correlated to the total nucleotide sequence of the housekeeping genes with a constitutively constant expression level. human genome (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Differences were considered statistically significant at a p-value of less than 0.05. qRT-PCR. The RT-qPCR was performed in triplicates. Previously synthesized cDNAs served as PCR templates. The amplification and Results detection of ALAS1, GAPDH, TBP, HPRT1 and B2M was performed with TaqMan Fast Universal Master Mix according to the Demographics and clinical parameters of the study cohort. manufacturers’s instructions (Applied Biosystems, Darmstadt, The study cohort consisted of 17 patients with median age of Germany). The final reaction volume of 10 μl consisted of 2× Master Mix (5.0 μl), TaqMan Gene Expression Mix (0.35 μl), aqua (2.65 μl) 67 years (range=51-77 years). Median preoperative and prediluted cDNA (2.0 μl). The cycle conditions were as follows: a concentration of prostate-specific antigen was 21.0 ng/ml step of the initial denaturation for 20 s at 95˚C followed by 40 cycles (range=3.2-154.0 ng/ml). Eight tumors were classified as of 5 s at 95˚C for template denaturation and 20 s at 60˚C for primer pT2, three as pT3 and one as pT4. Five tumors were clinically

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Figure 1. Expression levels of candidate housekeeping genes in metastatic and non-metastatic lymph nodes (LNs) from patients with prostate cancer. Ct: Cycle threshold numbers. Boxes: Lower and upper quartiles with medians. Whiskers, Ranges for Ct values.

classified as T1 following no surgical therapy (two with 0.4. Stepwise exclusion of the genes in order of the mean radiation therapy, one with hormonal therapy and one with stability led to determining TBP and HPRT1 as being the active surveillance). In terms of Gleason score, the median most consistently expressed housekeeping gene combination was 7 (range=5-9). Eight patients presented lymphatic disease in pelvic lymph nodes from patients with PC (M=0.17). (two patients of whom provided two positive lymph nodes each, and six provided one), nine patients presented pN0 (one Discussion patient provided two negative lymph nodes). None of the patients had visceral metastases (all cM0). The occurrence of pelvic LN metastases represents the first stage of PC dissemination, determining the switchover from Expression levels of candidate reference genes. The six potentially curative to palliative systemic disease. The status candidate housekeeping genes investigated had a wide range of of pelvic LN involvement is pivotal for predicting prognosis expression with Ct values ranging between 35 and 228 (Figure and assessment of further treatment. Since several groups 1). Using paired Student’s t-test, a significant difference in gene highlighted the prognostic role of the number and size of expression between malignant and non-malignant sample pairs lymphatic metastases as well as the extent of pelvic was observed for B2M. This gene was therefore excluded from lymphadenectomy (12-14), profound analysis of PC cell subsequent analysis by geNorm algorithm. biology in LN is of a prodigious scientific and clinical interest. In this context, gene expression profiling is expected Expression stability of candidate reference genes. After to shed light on the complex regulatory mechanisms during exclusion of B2M from further analysis, five candidate tumor cell spread. Detailed molecular analysis may facilitate reference genes (K-ALPHA-1, TBP, ALAS1, GAPDH and the detection of genes relevant to tumor dissemination and HPRT1) without significant differences in the expression potential therapeutic targets. level between metastatic and non-metastatic pelvic lymph qRT-PCR is widely applied for mammalian transcriptom nodes in PC patients were considered to be potentially analysis by amplifying specific nucleic acid target sequences suitable genes for use in normalization. In order to identify and quantification of the products (15). The analysis is the most stably expressed among the five candidate performed in real time during the log-phase of product housekeeping genes, validation with geNorm was performed. accumulation, and permits simultaneous assessment of The mean M values are presented in Figure 2. All genes multiple genes (16). This high-throughput method is provided high expression stability, with M values of less than characterized by its high degree of reproducibility, easy

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Figure 2. Selection of the most stably expressed reference genes for normalization in lymph nodes from patients with prostate cancer using geNorm algorithm. After exclusion of the unstable gene (B2M), the remaining five genes were analyzed. Y-axis: Average M value after exclusion of the least stably expressed gene and new calculation of the M value. Declining M values indicate increasing stability, with the combination of the two most stable genes being shown.

automation and its ability to reliably analyze even low probe resulting in varying mRNA expression even in cellular sub- volumes (10, 16). Similarly to other approaches for gene populations of the same pathological origin (22-25). Lin expression analysis, normalization is required in order to alluded to its very limited value for normalization of gene minimize the influence of inter-individual variability, amount expression with respect to the latest evidence (26). In of starting material, enzymatic properties and other contrast, Mori proposed GAPDH to be a reliable experimental conditions on the quantification results which denominator for gene expression in formalin-fixed paraffin- can induce misleading data. embedded PC tissues (18). Given the fact that ideal and Karge proposed cellular RNA, being expressed at steady universal control genes may probably not exist at all (10), levels independently from the sample, be amplified together Bustin recommended normalization be performed against a with target genes, serving as a reference for other RNA panel of housekeeping genes whose expression has been expression values to be normalized against (17). This should proven to be constant and unaffected by experimental ideally not vary in different tissues or cell types, be conditions, and to include this information in every expressed independently of cell-cycle status, stage of disease, publication (19). environmental conditions and development stage (18). To our best knowledge, the current study provides the first However, which is the ideal reference gene is still under evidence identifying potential housekeeping genes for gene debate. Controversy still exists even concerning which RNA expression analyses of LN metastasis in PC. In the first part type (total RNA, mRNA or rRNA) represents the most of our analysis, ALAS1, K-ALPHA-1, GAPDH, TBP and appropriate reference standard (10, 19). mRNAs of a number HPRT1 genes demonstrated similar transcriptional activity in of reference genes, for example GAPDH, ACTB, albumins, metastatic and non-metastatic LN and might theoretically tubulins, cyclophilin and rRNAs, have been recently serve as internal controls (depicted in Figure 1). In contrast, advocated as suitable candidates for PCR normalization and B2M mRNA expression was significantly down-regulated in meanwhile used in over 90% of cases. However, concern metastatic LNs. This demonstrated that B2M is unsuitable as about their reliability emerged as evidence and was provided a reference normalization in gene profiling studies of for variations in their expression in response to different metastatic and non-metastatic LNs. Therefore, this gene was factors (20, 21). excluded from further assessment. In the next step of the Even looking at GAPDH, the most frequently used analysis, we aimed to restrict the number of possible housekeeping gene in RT-PCR, astonishing discrepancies in reference genes and, most importantly, to distinguish a gene the published data can be found. GAPDH contributes to combination that would provide the most stable different cellular functions, such as nuclear RNA export, transcriptional activity and thereby represent an ideal DNA replication, DNA repair and cytoskeletal organization, reference standard for qRT-PCR of metastatic and non- phosphotransferase activity and pathological apoptosis, metastatic LN tissue from patients with PC. Thus, the

5246 Tsaur et al: Housekeeping Genes in Prostate Cancer geNorm software provided striking evidence for the genes 3 VanGuilder HD, Vrana KE and Freeman WM: Twenty-five years TBP and HPRT1 being most stably expressed in LNs from of quantitative PCR for gene expression analysis. Biotechniques patients with PC. 44: 619-626, 2008. The HPRT1 gene encodes for hypoxanthine phosphoribo- 4 Nolan T, Hands RE and Bustin SA: Quantification of mRNA using real-time RT-PCR. Nat Protoc 1: 1559-1582, 2006. syltransferase-1, an enzyme catalyzing the conversion of 5 Schmittgen TD and Zakrajsek BA: Effect of experimental hypoxanthine and guanine to their respective mononucleotides treatment on housekeeping gene expression: validation by real- thereby inducing purine salvage pathways (27). As purine time, quantitative RT-PCR. 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