A novel assay for exosomal and cell-free miRNA isolation and quantification.

The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality. To overcome these deficiencies, we developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared our assays with commercial techniques.

Copy number variations of circulating, cell-free DNA in urothelial carcinoma of the bladder patients treated with radical cystectomy: a prospective study.

The aim of the present study was to establish a rapid profiling method using multiplex ligation-dependent probe amplification (MLPA) and characterize copy number variations (CNV) in circulating, cell-free DNA (cfDNA) in 85 urothelial carcinoma of the bladder (UCB) patients treated with radical cystectomy (RC). MLPA was tested for the use of cfDNA extracted from serum and plasma by various commercial extraction kits. Eighteen probes served as reference to control denaturation, ligation and amplification efficiency.

Novel Technology for Enrichment of Biomolecules from Cell-Free Body Fluids and Subsequent DNA Sizing.

Circulating cell-free DNA (ccfDNA) is a promising diagnostic tool and its size fractionation is of interest. However, kits for isolation of ccfDNA available on the market are designed for small volumes hence processing large sample volumes is laborious. We have tested a new method that enables enrichment of ccfDNA from large volumes of plasma and subsequently allows size-fractionation of isolated ccfDNA into two fractions with individually established cut-off levels of ccfDNA length.

Specific DNA detection using antibody mediated fluorescence quenching.

First homogenous immunoassay for sequence-specific nucleic acid detection is developed. The assay bases on our finding that a fluorophore inserted into a DNA probe instead of one of the internal nucleotides may get protected from fluorescence quenching caused by an anti-fluorophore antibody, if the probe is hybridized with the target sequence. This ensures a positive signal in the antibody presence.

rapidSTRIPE H1N1 test for detection of the pandemic swine origin influenza A (H1N1) virus.

The rapidSTRIPE H1N1 test, based on a nucleic acid lateral-flow assay, has been developed for diagnosis of a swine-origin influenza A (H1N1) virus. This test is simple and cost-effective and allows specific detection of the S-OIV A (H1N1) virus from swab sampling to final detection on a lateral-flow stripe within 2 to 3 h.

Mutant-enriched PCR and allele-specific hybridization reaction to detect K-ras mutations in stool DNA: high prevalence in a large sample of older adults.

Background: Testing for mutant K-ras in stool has been proposed for the detection of pancreatic and colorectal cancer (CRC). Different analytical techniques have been developed, but studies of this biomarker in the general population are lacking. We investigated the prevalence and potential determinants of mutant K-ras in stool in a large sample of unselected older adults and assessed the association with colonoscopic findings.