MicroRNAs (miRNAs) are small RNA molecules, which act as post-transcriptional regulators of a gene expression, with important functions within the cell physiology. Whilst many authors have focused on the study of miRNA expression in physiological and pathological processes, various technical variables related to miRNA isolation have simultaneously emerged and the stability of the stored miRNA samples has been questioned. A robust method for RNA isolation is essential for reproducible results and miRNAs instability in the stored samples would make for an alarming situation for most expression studies. Here these issues are discussed and we investigate the stability of miRNAs isolated from clinical samples of B lymphocytes (chronic lymphocytic leukemia) by the most commonly utilized method based on a Trizol/TRI-Reagent solution (RNAs stored at -80 degrees C). To assess the stability of miRNAs, a Real Time-PCR analysis was performed for a panel of 29 miRNAs from a freshly isolated RNA sample and after 14 days storage at -80 degrees C. Furthermore, a Real Time-PCR analysis was repeatedly performed for a stored RNA sample over a period of approximately 10 months. We observed high stability of isolated miRNAs and respective cDNAs. The reproducibility and efficiency of the Trizol/TRI-Reagent isolation method was also tested and compared to the mirVana Isolation kit (Ambion) and RNeasy kit (Qiagen). In conclusion, Trizol/TRI-Reagent based isolation is a robust reproducible method, and obtained miRNA samples do not show any tendency to degradation when properly stored and handled.

• MeSH
• B-lymfocyty chemie   MeSH
• chronická lymfatická leukemie diagnóza   MeSH
• fenoly chemie   MeSH
• guanidiny chemie   MeSH
• lidé MeSH
• mikro RNA chemie   izolace a purifikace   MeSH
• odběr biologického vzorku MeSH
• polymerázová řetězová reakce MeSH
• reprodukovatelnost výsledků MeSH
• roztoky MeSH
• stabilita RNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• fenoly MeSH
• guanidiny MeSH
• mikro RNA MeSH
• roztoky MeSH
• trizol MeSH Prohlížeč

We compare two types of pancreatic carcinoma samples obtained by EUS-guided fine needle biopsy (EUS-FNB) in terms of the success rates and clinical validity of analysis of two most commonly investigated DNA/RNA pancreatic cancer markers, KRAS mutations and miR-21 expression. 118 patients with pancreatic ductal adenocarcinoma underwent EUS-FNB. The collected sample was divided, one part was stored in a stabilizing solution as native aspirate (EUS-FNA) and second part was processed into the cytological smear (EUS-FNC). DNA/RNA extraction was followed by analysis of KRAS mutations and miR-21 expression. For both sample types, the yields of DNA/RNA extraction and success rates of KRAS mutation and miRNA expression were evaluated. Finally, the resulting KRAS mutation frequency and miR-21 prognostic role were compared to literature data from tissue resections. The overall amount of isolated DNA/RNA from EUS-FNC was lower compared to the EUS-FNA, average yield 10 ng vs 147 ng for DNA and average yield 164 vs. 642 ng for RNA, but the success rates for KRAS and miR-21 analysis was 100% for both sample types. The KRAS-mutant detection frequency in EUS-FNC was 12% higher than in EUS-FNA (90 vs 78%). The prognostic role of miR-21 was confirmed in EUS-FNC (p = 0.02), but did not reach statistical significance in EUS-FNA (p = 0.06). Although both types of EUS-FNB samples are suitable for DNA/RNA extraction and subsequent DNA mutation and miRNA expression analysis, reliable results with clinical validity were only obtained for EUS-FNC.

• Klíčová slova
• EUS-FNA, KRAS, Pancreatic cancer, miR-21,
• MeSH
• biopsie tenkou jehlou pod endosonografickou kontrolou MeSH
• cytodiagnostika metody   MeSH
• DNA analýza   MeSH
• duktální karcinom slinivky břišní diagnóza   MeSH
• fixace tkání metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA analýza   MeSH
• mutace MeSH
• nádorové biomarkery analýza   MeSH
• nádory slinivky břišní diagnóza   MeSH
• odběr biologického vzorku metody   MeSH
• protoonkogenní proteiny p21(ras) genetika   MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA MeSH
• KRAS protein, human MeSH Prohlížeč
• mikro RNA MeSH
• MIRN21 microRNA, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• protoonkogenní proteiny p21(ras) MeSH