A phenol-free method for isolation of microRNA from biological fluids.

MicroRNAs (miRNAs) found in biological fluids such as blood and urine have been identified as promising biomarkers for many human disorders, including cancer, cardiopathies, and neurodegenerative diseases. However, circulating miRNAs are either encapsulated into vesicles or found in complexes with proteins and lipoproteins and, thus, require a special approach to their isolation. Acid phenol-chloroform extraction can solve this problem, but it is a labor-intensive procedure that relies heavily on the use of hazardous chemicals. Here we describe a fast and simple phenol-free protocol for miRNA isolation from biofluids. MiRNA is extracted from complexes with biopolymers by a high concentration of guanidine isothiocyanate combined with water/organic composition of solvents. Purification is finished using silica-based spin columns. Comparison of miRNA isolation from blood plasma and urine using the single-phase method and acid phenol-chloroform extraction by means of radioisotope spike-ins and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) showed similar performance of the two methods.

Cell-free and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content.

INTRODUCTION: Extracellular nucleic acids are found in human blood and cell culture medium as cell-free or being adsorbed at cell surface. In the last years, the circulating extracellular nucleic acids in blood were shown to be associated with certain diseases. Attempts are made to develop non-invasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. AREAS COVERED: This article reviews accumulating data regarding cell-free and cell-surface-bound extracellular nucleic acid nature and generation mechanisms. Their existence as a constituent of the naturally occurring complexes with proteins or membrane-bearing particles is discussed with regard to their homeostatic concentration and distribution in healthy donor blood which are significantly altered in cancer patients. Gene-target and whole-genome studies reveal significant differences in gene representation between extracellular DNA and genome DNA. Overrepresentation of regions with high transcription activity has led to proposal that extracellular DNA generation is strongly dependent on the parent genome functionality, which is associated with chromosome packaging and DNA methylation levels. EXPERT OPINION: Recent studies provide evidence of the circulating nucleome organization complexity indicating that discovery of extracellular DNA generation and circulation patterns in healthy condition and cancer is essential to enable the development of proper approaches for the selection of valid diagnostic markers.

A reliable method to concentrate circulating DNA.

Concentration of circulating DNA probes is required to increase the amount of DNA involved in subsequent study (by polymerase chain reaction, sequencing, and microarray). This work was dedicated to the comparison of five different methods used for concentration of DNA circulating in blood. Precipitation of circulating DNA with acetone in the presence of triethylamine provides minimal DNA loss, high reproducibility, and at least three times higher DNA yield in comparison with the standard ethanol protocol.

Methylation-specific sequencing of GSTP1 gene promoter in circulating/extracellular DNA from blood and urine of healthy donors and prostate cancer patients.

Hypermethylated promoters of cancer-related genes represent convenient targets for early cancer diagnosis and monitoring based on circulating/extracellular DNA (cir/exDNA) from human blood and urine. The frequency of detection of methylated tumor suppressor genes in plasma or urine samples is usually lower than in the samples of tumor tissue because of a low concentration of target DNA and potential polymorphism of cirDNA methylation. Sequencing of the methylated cir/exDNA of tumor suppression genes provides information about methylation of the cirDNA originating from the tumor cells, which is necessary for optimization of cancer diagnosis. In this work, by sequencing chemically converted cir/exDNA, we have studied the cytosine methylation profile of GSTP1 gene promoter (1001-1302, X08058) in the pool of cir/exDNA from the blood and urine of healthy men, prostate cancer (PCa) patients, and patients with benign prostatic hyperplasia (BPH). We demonstrated that the data on cir/exDNA methylation could be obtained from sequencing of the cir/exDNA from blood and urine. The DNA isolated from blood plasma and the eluates of blood cells and urine of each patient were characterized by the same methylation profile of the GSTP1 gene. The profile of GSTP1 gene methylation in the extracellular DNA of PCa patients differs from the profiles characteristic of healthy donors and patients with BPH.

Extracellular nucleic acids in cultures of long-term cultivated eukaryotic cells.

Investigation of the kinetics of nucleic acid release by HeLa (human cervical carcinoma cell line) and A431 (human squamous carcinoma cell line) cells is presented. The released DNA and RNA were shown to accumulate in culture medium and at the cell surface. A portion of cell surface bound RNA can be eluted with PBS/EDTA. Mild trypsin treatment is required for complete detachment of cell surface bound RNA and cell surface bound DNA. Electrophoretic analysis reveals characteristic patterns of cell-associated and free RNA and DNA molecules.