br RWPE ATCC CRL prostate epithelial
RWPE-1 (ATCC CRL-11609) prostate epithelial cell line, LNCaP clone FGC (ATCC CRL-1740) and VCaP (ATCC CRL-2876) prostate cancer cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). LNCaP was propagated in RPMI 1640 medium along with 10% FBS and 1% penicillin-strepto-mycin. VCaP was cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 1 mM sodium pyruvate. For RWPE-1, K-SFM with 5 ng/mL EGF and 50 μg/mL BPE were used for culture. All cell lines were incubated in a humidified Paclitaxel of 5% CO2 at 37 °C.
2.3. Urine sample preparation
Spot urine samples from 5 healthy male volunteers, first voided post-DRE urine from 5 BPH patients and from 5 PCa patients were provided by the Division of Urology, Department of Surgery, Faculty of Medicine Ramathibodi Hospital, Mahidol University. Diagnosis of pa-tients was made by histopathological analysis after prostate biopsy subsequently. PCa patients were identified with positive biopsy. This study was approved by the Committee on Human Rights Related to Research Involving Human Subjects, Faculty of Medicine Ramathibodi Hospital, Mahidol University (MURA2016/34). Written informed con-sent was obtained from all subjects in the study. The urine samples were collected and then centrifuged at 4 °C, 3000 rpm for 10 min to separate the cells. Cell pellets were washed twice by PBS, pH 7.0. Finally, TRIzol reagent (Invitrogen, CA, USA) was added to the sediments which were then stored at −20 °C until RNA isolation.
2.4. RNA extraction and cDNA synthesis
Total RNA was isolated from the cell pellets of urine as well as from cell lines with TRIzol reagent. 500 μg of total RNA was converted to cDNA using RevertAid First Strand cDNA synthesis kit according to the manufacturer's instructions and stored at −20 °C until use.
2.5. RT-PCR and PCR purification
PCA3 amplification was performed using a thiol-labeled or un-labeled forward primer, and an unlabeled reverse primer. The se-quences of primers are shown in Table 1. Thiolated PCR products contained thiol-labeling at the 5′ end. PCR was carried out in a 50 μL reaction mixture containing 0.05 μg of cDNA template, 0.5 μM of each primer, 1× PCR reaction buﬀer, 0.2 mM dNTPs and 2.5 U i-Taq DNA polymerase. Of note, to amplify PCA3 from cDNA of clinical samples, 2.5 U Phusion high-fidelity DNA polymerase was applied in the PCR reaction instead of i-Taq DNA polymerase. The cycling procedures were
Fig. 3. Specificity analysis of AuNP-based colorimetric assay. (A) PCR products of PCA3 (upper gel) and GAPDH (lower gel) analyzed by 1.5% agarose gel elec-trophoresis. Amplicon sizes of PCA3 and GAPDH were 838 bp and 106 bp, respectively. Lane M, 100 bp ladder; Lanes 1–3, PCR products from LNCaP, VCaP and RWPE-1, respectively; Lane NTC, no-target control. (B) Visual detection by the naked eye of AuNPs mixed with corresponding thiolated PCA3 products from LNCaP, VCaP and RWPE-1 in wells 1–3, respectively. (C) UV–vis spectral analysis of the AuNP solutions containing PCR products and NTC. The order of the reactions is similar to gel electrophoresis. A520/A640 ratio determined by UV–vis spectrophotometer. Each bar represents the mean ± SD obtained from three experiments. The statistical analysis utilized the t-test. *P-value < .05 compared with NTC.
set for 5 min at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at 50 °C, and 90 s at 72 °C, and finally extension 10 min at 72 °C. After amplification, 10 μL PCR products were analyzed by agarose-gel elec-trophoresis. The PCR amplicon size was 838 bp. The rest of PCR pro-ducts were purified and eluted using gel and PCR clean-up kit according to the manufacturer's instructions. GAPDH gene, a housekeeping gene, was used as a control to check cDNA quality. To amplify GAPDH, the cycling procedures were performed as followed: initial denaturation at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, and a final ex-tension step of 5 min. The sequences of primers are shown in Table 1. The PCR amplicon size was 106 bp.