We analyzed the effect of hemolysis on microRNA profi le of blood plasma. It was found that hemolysis of ~0.05% erythrocytes in a sample signifi cantly affected the concentration of 9 microRNA: hsa-miR-486-5p, hsa-miR-16-5p, hsa-miR-451a, hsa-miR-106a-5p, hsa-miR-17-5p, hsa-miR-93-5p, hsa-miR-20a-5p, hsa-miR-107, and hsa-miR-20b-5p. The effect of hemolysis on plasma content of miR-17 family microRNA was demonstrated.
Peripheral blood plasma profiles of circulating microRNA expression were analyzed in patients with prostatic cancer and benign hyperplasia. In prostatic cancer, significant increase in hsa-miR-619-5p and hsa-miR-1184 microRNA expression and significant decrease in hsalet-7b-5p and hsa-let-7c-5p microRNA expression were observed. The role of the relationship between the microRNA expression and the activities and functions of host genes with introns encoding these microRNA is discussed.
Missense mutations in the VHL gene during sporadic clear cell renal cell carcinoma were studied to evaluate their localization in relation to functionally important motifs of the VHL protein. Somatic mutations were identified in 124 of 307 samples. All missense mutations in the α-domain were localized in the binding site for elongin C. Substitutions in the β-domain (77%) were found in the HIF-binding site. Five missense mutations were absent in these sites, which illustrates their role in VHL protein formation or suppressor function of other protein cofactors. Mutation c.392A→T (p.N131I) was identified for the first time. Our results hold much promise to estimate the boundaries of functionally important sites in the VHL suppressor gene and contribute to the interpretation of a pathogenic role of mutations in direct DNA diagnostics.
Molecular genetic analysis of KRAS, NRAS, and BRAF genes was carried out in order to develop an optimal algorithm for detection of minor mutations. We analyzed 35 melanoma and 33 colorectal cancer specimens. Frequent G12D/V/A/C/S mutations were detected in KRAS. The most frequent BRAF mutation in melanoma was V600E, the percentage of rare mutations is significant for DNA diagnosis (24%). Identification of rare BRAF mutations 1790C→G (L597R), 1798_1799delinsAA (V600K), 1798_1799delinsAG (V600R), and 1799_1800delinsAA (V600E) and NRAS mutation 38G→T (G13V) was possible only by Sanger sequencing. The combination of real-time PCR and sequencing can improve analysis sensitivity and ensure concordance of the tested loci with the international recommendations.
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