Evaluation of the efficacy of various reagents in improving microRNA extraction.

BACKGROUND: MicroRNA has received considerable attention in the clinical context, and attempts are being made to use microRNA in clinical diagnosis. However, adequate quantities of microRNA required for analysis are challenging to isolate. We tested the effect of various reagents in improving microRNA extraction and compared their efficacy to that of a commercially available extraction kit (HighPure miRNA isolation kit, Roche). METHODS: We used the synthetic oligonucleotide miR-21 and formalin-fixed, paraffin-embedded (FFPE) tissue sections from colon cancer samples ( n = 10). We tested increasing volumes (100-600 µL) of 1,4-dioxane, 2-butanol, 2-propanol, acetonitrile, polyethylene glycol (PEG) 600, PEG 1000, PEG 1540, PEG 2000, tetraethylene glycol dimethyl ether (TDE), and tetrahydrofuran, instead of the binding enhancer solution provided in the kit. MiR-21 analysis was performed via stem-loop RT-qPCR using Universal ProbeLibrary probe (Roche). RESULTS: The optimum amount of each enhancement solution was 200-500 µL. We obtained ΔCp values of optimum additional volume for each solution from 1.04 to 2.50 and compared these with those obtained using the commercially available kit. PEG 1540 and 2000 produced superior reactivity with minimal addition. For FFPE tissue samples, addition of the enhancement solutions PEG 1540 and 2000 resulted in mean crossing point values of 18.15 ± 2.26 and 17.73 ± 3.26, respectively. We obtained a crossing point value of 20.56 ± 4.26 (mean ± SD) using the commercially available kit. CONCLUSIONS: The tested enhancer reagents, which are relatively readily available and easy to use, can improve microRNA extraction efficacy of a commercially available kit.

Improved Target Cell Selection and Counting Method for UroVysion Fluorescence in Situ Hybridization.

BACKGROUND: The UroVysion Bladder Cancer Kit requires morphological analysis of 4', 6-diamino-2-phenylindole (DAPI)-stained nuclei to identify target cells for fluorescence in situ hybridization (FISH) signals. Reproducibility and efficiency of target cell selection and counting was evaluated by combining immunofluorescence staining of cytokeratin 7 (CK7) and proliferating cell nuclear antigen (PCNA) with DAPI staining. METHODS: The reactivities to CK7, PCNA, and DAPI were compared between those for different ratios of T24 human bladder carcinoma cells and of cells from the urine of five healthy subjects. Two technicians independently performed five replicate cell counts of urine samples from four bladder cancer patients and one healthy subject. RESULTS: The positive staining rates for CK7 and PCNA were similar to DAPI, but our method showed enhanced inter-observer repeatability and reduced operating time for signal counting. CONCLUSIONS: Our proposed method showed better reproducibility and lesser operational time for signal counting than the DAPI method alone.

Detection of bladder cancer by measuring CD44v6 expression in urine with real-time quantitative reverse transcription polymerase chain reaction.

OBJECTIVE: To examine urinary CD44v6 total ribonucleic acid (RNA) expression in patients with bladder cancer using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and evaluate its potential as a novel marker of bladder cancer. METHODS: We used the bladder cancer cell line T24 and determined CD44v6 expression in cancer cells using in situ hybridization and immunohistochemistry. Subsequently, we obtained urine samples from 21 patients with bladder cancer and 25 patients without bladder cancer (controls). We extracted total RNA from the urine samples, measured CD44v6 total RNA expression in both groups using qRT-PCR, and compared the expression between groups. We also compared the sensitivity, specificity, and concordance rate between CD44v6 total RNA expression analysis by qRT-PCR and cytologic analysis, UroVysion fluorescent in situ hybridization, bladder tumor antigen identification, and nuclear matrix protein 22 measurements. RESULTS: We observed increased CD44v6 expression in bladder cancer cells using in situ hybridization and immunohistochemistry. CD44v6 total RNA expression was significantly higher in the urine samples of patients with bladder cancer than in those of controls. We calculated the cutoff value from the receiver operating characteristic curve and obtained sensitivity and specificity values of 85.7% and 72.0%, respectively, for qRT-PCR analysis. CONCLUSION: Our results suggest that CD44v6 total RNA levels in urine can serve as a potential noninvasive biomarker of bladder cancer.

Use of task-specific ionic liquid for selective electrocatalytic fluorination.

Highly selective indirect anodic fluorination of organic compounds was successfully carried out for the first time by using a task-specific ionic liquid of iodoarene as a mediator in ionic liquid hydrogen fluoride salts.