A complete diagnostic procedure was developed that allows single molecules of mRNA AML1-ETO to be detected in samples of whole blood and bone marrow of the leukemia t(8;21)(q22;q22) patients. The procedure includes: a method for preservation of biological samples ensuring the RNA integrity; an improved method for isolation of RNA from the unfractionated whole blood and bone marrow; an optimized reverse transcription; and the use of nanocolonies for detection and enumeration of RNA target molecules. The developed procedure is the first one that provides for determination of the absolute titer of an RNA target without reference to a control (housekeeping) gene, and significantly increases sensitivity, precision and reliability of detection of the minimal residual disease at a leukemia associated with known chromosomal translocation.
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Identifying RNA recombination events and non-covalent RNA-RNA interactions with the molecular colony technique.
Methods Mol Biol
June 2015
Institute of Protein Research of the Russian Academy of Sciences, Institutskaya st., 4, Pushchino, Moscow Region, 142290, Russia.
Molecular colonies (also known under names nanocolonies, polonies, RNA or DNA colonies, PCR colonies) form when nucleic acids are amplified in a porous solid or semi-solid medium, such as a gel, which contains a system for the exponential multiplication of RNA or DNA. As an individual colony comprises many copies of a single molecule (a molecular clone), the method can be used for the detection, enumeration, and analysis of individual DNA or RNA molecules, including the products of such rare events as RNA recombinations. Here we describe protocols for the detection of RNA molecules by growing colonies of RNA (in a gel containing Qβ replicase, the RNA-dependent RNA polymerase of phage Qβ) or cDNA (in a gel containing the components of PCR), and visualizing them by hybridization with fluorescent probes directly in the gel, including in real time, or by hybridization with fluorescent or radioactive probes followed by transfer to a nylon membrane.
View Article and Find Full Text PDFNanocolonies and diagnostics of oncological diseases associated with chromosomal translocations.
Biochemistry (Mosc)
December 2010
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
This paper reviews chromosomal abnormalities observed in oncological diseases, the history of discovery of chromosomal translocations (a widespread type of chromosomal abnormalities), and statistical data showing a correlation between translocations and emergence of oncological diseases (in particular leukemia). The importance of detection of minimal residual disease (MRD) in treatment of leukemia associated with translocations is discussed along with methods of MRD diagnosis, followed by description of a novel diagnostic procedure for the detection of single chimeric mRNA molecules serving as MRD markers. This procedure includes a number of improvements, of which the most important is the use of a PCR version of the method of nanocolonies (other names are molecular colonies, polonies) that provides for the determination of the absolute titer of RNA tumor markers, excludes false positive results in the detection of chimeric molecules, and significantly exceeds other methods in the sensitivity of MRD detection.
View Article and Find Full Text PDFA complete diagnostic procedure was developed that allows single molecules of mRNA AML1-ETO to be detected in samples of whole blood and bone marrow of the leukemia t(8;21)(q22;q22) patients. The procedure includes: a method for preservation of biological samples ensuring the RNA integrity; an improved method for isolation of RNA from the unfractionated whole blood and bone marrow; an optimized reverse transcription; and the use of nanocolonies for detection and enumeration of RNA target molecules. The developed procedure is the first one that provides for determination of the absolute titer of an RNA target without reference to a control (housekeeping) gene, and significantly increases sensitivity, precision and reliability of detection of the minimal residual disease at a leukemia associated with known chromosomal translocation.
View Article and Find Full Text PDFNanocolonies: detection, cloning, and analysis of individual molecules.
Biochemistry (Mosc)
December 2008
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Nanocolonies (other names molecular colonies or polonies) are formed upon template nanomolecule (DNA or RNA) amplification in immobilized medium with efficient pore size in the nanometer range. This work deals with the principle, invention, development, and diverse nanocolony applications based on their unique abilities to compartmentalize amplification and expression of individual DNA and RNA molecules, including studying reactions between single molecules, digital molecular diagnostics, in vitro gene cloning and expression, as well as identification of the molecular cis-elements including DNA sequencing, analysis of single-nucleotide polymorphism, and alternative splicing investigation.
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