Improved sensitivity and specificity for prostate cancer diagnosis based on the urine PCA3/PSA ratio acquired by sequence-specific RNA capture

Abstract

Abstract. Prostate cancer antigen 3 (PCA3) is a non-coding RNA fragment that is overexpressed in prostate cancer cells. However, the clinical applications of PCA3 are highly limited due to the instability of RNA and the lack of reliable and efficient RNA extraction and purification methods. Thus, in the present study, we compared three different methods of RNA extraction to further confirm the higher yield of commercial magnetic beads with poly-T functionalization and a capturer strand. The current protocols for RNA extraction of i) the phenol-chloroform method, ii) the affinity column method and iii) magnetic beads with poly-T functionalization and a capturer strand were applied separately for RNA extraction in urine samples. Reverse transcription-quantitative polymerase chain reaction was performed to evaluate the yield of the three methods of RNA extraction. Furthermore, 52 urine samples after prostate massage from patients suspected of a diagnosis of prostate cancer were collected. The Mag-Cap method and RT-PCR were applied to obtain the PCA3 score. The clinical value of the PCA3 score was investigated by comparison with the pathology of the prostate biopsy. The yield of the Mag-Cap method was higher than that of the phenol-chloroform method and commercial kits. Thirty-four patients were pathologically diagnosed with prostate cancer and 18 with benign prostatic hyperplasia (BPH). It was confirmed that the median PCA3 score was higher among the prostate cancer patients than those with benign disease (53.5 vs. 17, p=0.000). A sensitivity of 82.4% and a specificity of 77.8% were obtained when the cut-off value for the PCA3 score was 28.5. The Mag-Cap method was found to be more efficient for RNA extraction. The urinary PCA3 score is a promising method for prostate cancer screening, detection and diagnosis, and has the potential to reduce unnecessary prostate biopsies.

The long non-coding RNA PCA3: an update of its functions and clinical applications as a biomarker in prostate cancer

“…As a typical RNA molecule, which have sequences that contain the active RNA degradation systems and therefore present defects in processing, folding, or assembly with proteins, being rapidly degraded by the surveillance machinery, PCA3 is unstable and its capture and preservation needs to be accurate. Aiming to circumvent this limitation, some approaches have been tested in order to improve higher yields of RNA extraction, such as commercially available magnetic beads (Mag-Cap), phenol-chloroform, affinity columns or Mag-Cap for RNA extraction in urine samples [55].…”

Diagnostic significance of urinary long non-coding PCA3 RNA in prostate cancer

“…However, the score of urinary PCA3 RNA versus PSA RNA showed a more favorable benefit than the single copy of PCA3 RNA, PSA RNA and serum tPSA for diagnosis of CaP. Meanwhile, our method achieved very satisfactory sensitivity and specificity in test [ 10 , 11 ]. Notably, the urine sample was collected after the DRE operation and without conducting prostatic massage which is different from the reported method [ 12 , 13 ].…”

New Uses for the Personal Glucose Meter: Detection of Nucleic Acid Biomarkers for Prostate Cancer Screening

“…In this sense, even if upregulated in prostate cancer, the concentration of PCA3 in urine is scarce, and it may be present at a femtomolar level. Consequently, it forces the implementation of a previous preconcentration step that would be compatible with magnetic beads technology [ 37 ]. Likewise, to exclude any nonspecific variation, levels of PCA3 should be normalized with an internal control of stable expression, which demands the development of a parallel test for this latter.…”