sectioned. Representative serial sections (7 mm-thick) were E&H stained (Figure 1). Right after the last section (the so-called ‘‘pre- RNA’’ frozen section), 10–12 frozen sections at 30 mm-thickness, for a total of about 20 mg of tissue, were collected and stored at 280uC for RNA extraction. Then the cryostat was set back to 7 mm thickness and ‘‘post-RNA’’ frozen section were cut for E&H staining. This ‘‘sandwich’’ procedure (Figure 1) rendered 2 frozen sections with a thickness of 7 mm named ‘‘pre-RNA’’ and ‘‘post- RNA’’. They were both used by the pathologist for the pathological classification of the 300–350 mm-thick tissue speci- men contained in between. This material was then used for RNA extraction. Classification by the pathologist of the pre- and post- RNA sections was two-fold: presence or absence of cancer. No Gleason grade or score was given on these frozen sections because morphology was not preserved by fixation. Thank to this procedure we gained as much morphological information as possible from the tissue specimen that was homogenized for molecular analysis. For RT-qPCR analysis we only used CaP specimens with a cancer cell content covering at least 5% of the whole section area. Only specimens with no PIN lesions or tumour invasion were used as controls (benign). We also collected clinically relevant parameters: age of the patient, total serum PSA before surgery. Final Gleason score of the tumour was assessed after formalin fixation and pathological examination of the whole gland. RNA extraction Total RNA was extracted from 20 mg snap-frozen human tissue specimens using RNA-fast (Molecular Systems, San Diego, CA) according to the manufacturer protocol. After spectrophotometric quantification, 2-mg aliquots were routinely electrophoresed on a 1% agarose gel to check quality and integrity as previously described [3]. Retrotranscription Two mg of total RNA from each sample was primed with 50 ng of random hexamers (Invitrogen) and incubated at 42uC for 60 min in a 30 mL (final volume) reaction mixture containing 50 mM tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTPs and 300 units of Superscript II Reverse Transcriptase (Invitrogen). Real-time PCR amplification One mL of each cDNA preparation was PCR-amplified using the set of primers described below. PCR conditions were: 95uC for 15 min and then 95uC for 30 s, 60uC for 30 s and 72uC for 30 s repeated for 40 cycles, then 95uC for 30 s and 55uC for 30 s. Melting curves were obtained. RT-qPCR was performed on the DNA Engine Opticon 4 machine (MJ Research, Wathman, MA, USA) using a 26 SYBR Green customized master mix (Biodiversity s.r.l., Brescia, Italy). Primers: References GAS1 DIR: 59- CGC TGA GCC GCT ACC TGA -39 REV: 59- CTT GGG CAT AGC CAG CAT GT -39 1. Pelzer AE, Tewari A, Bektic J, Berger AP, Frauscher, et al. (2005) Detection rates and biologic significance of prostate cancer with PSA less than 4.0 ng/mL: observation and clinical implications from Tyrol screening project. Urology 66: 1029–33. 2. Huppi K, Chandramouli G (2004) Molecular profiling of prostate cancer. Curr Urol Rep 5: 45–51. 3. Bettuzzi S, Davalli P, Astancolle S, Carani C, Madeo B, et al. (2000) Tumor progression is accompanied by significant changes in the levels of expression of polyamine metabolism regulatory genes and Clusterin (Sulfated Glycoprotein 2) in human prostate cancer specimens. Cancer Res 60: 28–34. Gene Signature H3 DIR: 59- CAG GAG GCT TGT GAG GCC TA -39 REV: 59- AGC TGG ATG TCT TTG GGC AT -39 SSAT DIR: 59 - GGT TGC AGA AGT GCC GAA AG -39 REV: 59- GTA ACT TGC CAA TCC ACG GG -39 CLU DIR: 59- TGA TCC CAT CAC TGT GAC GG -39 REV: 59- GCT TTT TGC GGT ATT CCT GC -39 ODC DIR: 59- AGA CCT TCG TGC AGG CAA TC -39 REV: 59- AGG AAA GCC ACC GCC AAT AT -39 AdoMetDC DIR: 59- CAT CAC TCC AGA ACC AGA AT -39 REV: 59- TAA CAA ACA AGG TGG TCA CA -39 OAZ DIR: 59- CCT CCA CTG CTG TAG TAA CC -39 REV: 59- GAA AGA TTG TGA TCC CTC TG -39 GAPDH DIR: 59- AAC CTG CCA AAT ATG ATG AC -39 REV: 59- TTG AAG TCA GAG GAG ACC AC -39 HMBS DIR: 59- TGA AAT CAT TGC TAT GTC CA -39 REV: 59- ATG TTC AAG CTC CTT GGT AA -39 PGK1 DIR: 59- CTT TCA TGT GGA GGA AGA AG -39 REV: 59- TAG CTT GGA AAG TGA AGC TC -39 RT-qPCR data analysis Analysis of the relative expression of target genes was performed with REST 2005 (Relative Expression Software Tool) BETA V1.9.9. [26]. Two housekeeper genes phosphoglycerate kinase 1 (PGK1) and hydroxy-methyl-bilane synthetase (HMBS) were used in the study. Also primers efficiency was calculated and used for REST analysis. Statistical Analysis Raw Ct data were normalized and transformed in DCt by using the geometric mean of both housekeepers (PGK1 and HMBS). Normalized DCt data were used for classification. The NN classifier was used in order to discriminate CaP from benign tissue. The 10-fold crossvalidation procedure was used for final validation in order to reduce the underestimation of the misclassification error due to over-fitting. All the possible combinations of the 8 genes, several numbers of neighbours and different distances between sample profiles (Euclidean distance, Correlation-based distance, Mahalanobis distance) were considered in the analysis. The same procedure was used for sub-classification of CaP specimens into several dichotomies: patients whose age was #65 versus .65; total PSA value at prostatectomy ,10 versus $10; final Gleason score on fixed whole prostate gland ,7 versus $7. Author Contributions Conceived and designed the experiments: SB. Performed the experiments: FR LB SB. Analyzed the data: FR LB PC DR. Contributed reagents/ materials/analysis tools: PC ML SF PC AC. Wrote the paper: FR DR SB. 4. Bettuzzi S, Scaltriti M, Caporali A, Brausi M, D’Arca D, et al. (2003) Successful prediction of prostate cancer recurrence by gene profiling in combination with clinical data: a 5-year follow-up study. Cancer Res 63: 3469–72. 5. Konishi H, Steinbach G, Terry N, Lee J, Dubin J, et al. (1996) Histone H3 messenger RNA in situ hybridization correlates with in vivo bromodeoxyuridine labeling of S-phase cells in rat colonic epithelium. Cancer Res 56: 434–7. 6. Del Sal G, Ruaro M, Philipson L, Schneider C (1992) The growth arrest-specific gene, GAS1, is involved in growth suppression. Cell 70: 595–607. 7. Bettuzzi S, Hiipakka R, Gilna P, Liao S (1989) Identification of an androgen- repressed mRNA in rat ventral prostate as coding for sulphated glycoprotein 2 by cDNA cloning and sequence analysis. Biochem J 257: 293–6. PLoS ONE | www.plosone.org 7 October 2008 | Volume 3 | Issue 10 | e3617