Method of obtaining overall fraction of extracellular nucleic acids from blood

FIELD: chemistry.

SUBSTANCE: method includes collecting blood in an anticoagulant solution, adding an equal volume of an elution buffer containing 1 M NaCl, 0.2 M NaHCO3 (pH 9.3), 20 mM EDTA or a buffer containing 1 M NaCl, 20 mM EDTA. The obtained mixture is incubated at room temperature for 4-6 minutes while stirring, followed by separating the collected blood into plasma and a blood cell fraction by centrifuging and collecting the supernatant fluid, from which the overall fraction of extracellular nucleic acids (exNA) is extracted.

EFFECT: use of the invention shortens the duration of obtaining an overall fraction of exNA, increases the output of blood exNA, increases the sensitivity of detecting market NA, which are specific for cancerous diseases and pregnancy pathologies.

3 cl, 3 tbl, 4 ex

 

The invention relates to the field of diagnostic medicine, namely to molecular genetic diagnosis, and is aimed at improving diagnostic efficiency of analytical systems based on the use of circulating extracellular nucleic acids (freely circulating in plasma and associated with the surface of blood cells).

Urgent task in diagnostic medicine is timely and accurate identification of patients by specific sequences of nucleic acids associated with various diseases. Especially intensive research in the field of test development based on analysis of circulating extracellular nucleic acids in blood (VNC) since the blood is in direct contact with all organs/tissues, and a number of processes, such as pregnancy, disruption of cells and tissues, the development of tumors is accompanied by a change in the composition of circulating nucleic acids in blood even at the earliest stages of development of these processes, in particular when the tumor is difficult/impossible to localize physical methods (1).

It has been previously shown that the use in the diagnosis VNC associated with the surface of blood cells of nucleic acids (spkn), along with the widely used VNC, freely circulating in blood plasma (2), allows to exis�tively improve the sensitivity of diagnostic systems (3).

Known method of separating vjnk associated with the surface of blood cells (4), comprising the following stages: collection of blood by gravity into tubes with phosphate buffer (10 mm) and EDTA (50 mm EDTA pH 7.5); the separation of blood into plasma and cellular fraction by centrifugation (20 min at 400 g) separation of the cellular fraction of blood leukocytes and erythrocytes; obtaining DNA associated with the surface of red blood cells and DNA associated with the surface of leukocytes by means of incubation of the cells with phosphate elution buffer (5 min incubation with subsequent precipitation of the cells by centrifugation for 20 min at 400 g); obtaining vjnk from eritrotsitarnoj and leukocyte fractions by means of incubation of the cells with trypsin solution and then trypsin inhibitor (5 min incubation with trypsin solution, addition of an inhibitor of trypsin, stirring the solution, the deposition of cells by centrifugation for 20 min at 400 g). Vjnk was isolated from blood plasma and the obtained eluates from the surface of blood cells through fine glass.

The disadvantages of this method are time-consuming procedure of obtaining fractions containing vjnk blood; and the use of trypsin in the process of obtaining vjnk that requires accuracy in the execution of protocols and control over the activity of the drug.

Known method of separating vnrc associated with the surface of kletochnoi (5), comprising the following stages: collection of blood by gravity into tubes with phosphate buffer (10 mm) and EDTA (50 medta pH 7.5); the separation of blood into plasma and cellular fraction by centrifugation (20 min at 400 g); obtaining vnrc blood cells through incubation of the cells with phosphate elution buffer (5 min incubation followed by centrifugation for 20 min at 400 g); obtaining vnrc blood cells through incubation of the cells with trypsin solution and then trypsin inhibitor (5 min incubation with trypsin solution, addition of an inhibitor of trypsin, mixing and deposition of cells by centrifugation for 20 min at 400 g). The allocation vnmc from plasma was performed on glass-fibre filters.

This method had previously listed disadvantages, the total time to obtain fractions containing vnrc is 2 hours and 42 minutes (time is designed to handle 2 blood samples with a volume of 8 ml), then the method cannot be automated.

Closest to the claimed method prototype is a method of separating vjnk from blood, comprising the following stages: collection of blood in vacutainers; the separation of blood into plasma and cellular fraction by centrifugation (20 min at 400 g); obtaining vjnk as a result of sequential processing of blood cells phosphate elution buffer (5 min incubation followed by centrifugation for 20 min at 400 g), then the processing of blood cells with a solution of trypsin (5 min incubation with trypsin solution, addition of an inhibitor of trypsin, mixing and deposition of cells by centrifugation for 20 min at 400 g). Vjnk blood from the resulting supernatants were isolated on glass fiber filters by means of a set of "Blood DNA isolation Kit (BioSilica Ltd, Russia) (6).

The disadvantages of this method are the complexity and duration of obtain fractions containing vjnk blood: total time to obtain fractions containing vjnk is 2 hours and 42 minutes. In addition, the known method requires constant monitoring of the effectiveness protease processing. The method includes many steps, requires constant fixation volumes, it is extremely difficult to automate. When two fractions are analyzed vjnk: separately - vjnk blood plasma and separately vjnk from the cellular fraction of blood, which doubles the cost allocation vjnk and PCR analysis of the samples.

The object of the invention is to provide a simple, fast and reproducible method for obtaining total fraction VNC blood containing simultaneously VNC from blood plasma and VNC from the cellular fraction of blood for later use VNC as a diagnostic material.

Effect: simplification, reduction in the duration of the method and increase the yield of VNC.

The task is achieved by the proposed method is as follows.

Undertake data collection, crovari the help of a tube or other tubes for the collection and preservation of blood (for example, Cell-Free DNA BCT, Streck, USA). To the aliquot of blood is added with an equal volume of elution buffer (DDB) containing 1 M NaCl, 0.2 M NaHCO3(pH 9,3), 20 mm EDTA or buffer containing 1 M NaCl, 20 mm EDTA, the mixture was incubated at room temperature for 4-6 minutes, stirring gently. The mixture of blood and elution buffer DDB separated into supernatant and cell fraction using double centrifugation (5 min at 1000 g for 5 minutes at 1,200 g). The resulting supernatant contains the total fraction VNC, including VNC plasma and VNC cell fraction. Further, from the supernatant VNC isolated by any suitable method of extraction of nucleic acids, namely by a method based on extraction with phenol-chloroform; guanidine-phenol method; method of extraction of nucleic acids using a fine glass or glass-fiber filters in the presence chaotropic salts. Quantitative analysis wnnk blood also carried out by any known method in the study of nucleic acids, such as quantitative PCR and RT-PCR; methyl-specific quantitative PCR method for measuring the concentration of TC using fluorescent dyes (Hoechst 32258, SYBR Green II, DAPI, etc.).

The defining difference of the proposed method in comparison with the prototype is that the sample of blood is added with an equal volume of elution buffer containing 1 M NaCl, 0.2 M NaHCO 3(pH 9,3), 20 mm EDTA or buffer containing 1 M NaCl, 20 mm EDTA, the mixture was incubated at room temperature for 4-6 minutes with stirring, which results in a total fraction containing VNC from blood plasma and VNC from cellular fractions of the blood, which in turn allows you to simplify the method, to reduce its duration and increase the yield of nucleic material, which allows to increase the sensitivity of detection of marker DNA sequences and RNA.

The proposed method has the following advantages compared with prototype:

- ensures lysis of fewer blood cells than the prototype, and is resistant to deviations from the regulations no more than 20%, making it suitable for use in clinical laboratories for performing routine research and automates the process;

- reduces the time of obtaining the total fraction VNC more than 4 times (2 hours 42 minutes to 34 minutes);

- allows up to 4 to reduce the total volume of the resulting fractions (from 44.5 ml to 12 ml), which reduces the complexity, reduce the costs of reagents for allocation of the NC, which in turn reduces the cost of the tests based on the use of VNC blood.

The invention is illustrated by the following examples of a specific implementation method.

Example 1

In group h�orovich donors (n=10) were conducted, the allocation of total fraction vjnk, obtained by the method-prototype (6) and the proposed method. All healthy volunteer donors was taken by the blood through the tube with anticoagulant. The collected blood was divided into 2 equal parts, one part was processed in three stages according to the Protocol prototype, and the remaining part is processed in one stage of the inventive method: the aliquot of blood was added an equal volume of elution buffer (DDB) containing 1 M NaCl, 0.2 M NaHCO3(pH 9,3), 20 mm EDTA and incubated the mixture at room temperature for 4 minutes, gently stirring the mixture of blood and elution buffer DDB was separated into supernatant and cell fraction using double centrifugation (5 min at 1000 g for 5 minutes at 1,200 g), and the obtained supernatant vjnk was isolated by using a fine glass in the presence chaotropic salts (4). The DNA concentration was determined using TaqMan-PCR for LINE-1 repeats (7).

The results of the study are presented in Table 1. As can be seen from table 1, using the proposed method of obtaining the total fraction vjnk blood allows to increase the output vjnk on average, 20,07%. While saving time is 2 hours and 8 minutes.

This example illustrates the use of the developed method in clinical laboratories using production methods with high reproducibility.

Example 2

In the group of patients with lung cancer (n=10) were conducted, the allocation of total fraction vjnk obtained using the prototype method (6) and the proposed method. All host in a study of people was taken by the blood through the tube. The collected blood was divided into 2 equal parts, one part was processed in three stages according to the Protocol prototype, and the second part was treated in one stage using the proposed method, namely: to the aliquot of blood was added an equal volume of elution buffer (DDB) containing 1 M NaCl, 20 mm EDTA and incubated the mixture at room temperature for 6 minutes, gently stirring the mixture of blood and elution buffer DDB was separated into supernatant and cell fraction using double centrifugation (5 min at 1000 g and 5 minutes at 1200 g), and from the resulting supernatant vjnk was isolated using sets of Biosilica (LLC Biosilica, Russia). Concentration ofwholesale DNA was determined using methyl-specific SYBR-Green PCR for the methylated fragment of the gene RARbeta2 (8).

Table 2 presents the concentration of the methylated form of the gene RARβ2 in the composition vjnk in the blood of patients with lung cancer, where the Protocol is 1 - method-prototype. Protocol 2 - the proposed method.

As can be seen from table 2, using the proposed method of obtaining the total fraction vjnk from the blood cancer b�found can significantly increase the output ofwholesale vjnk an average of 58%, the time required to execute the proposed method reduces more than 4 times (2 hours 42 minutes to 34 minutes).

Example 3

In the group of pregnant RhD-negative women waiting RhD-positive child (n=20), was a comparative study of the concentrations of total fraction vjnk and genetic marker RH supplies (RHD) in samples obtained using the prototype method (6) and the proposed method similarly to example 1. In all the patients was taken by the blood through the tube. The collected blood was divided into 2 equal parts, one part was processed according to the Protocol prototype to the stage of the use of elution buffers (receive only the samples of blood plasma), and the second part was processed using the proposed method similarly to example 1, with vjnk from the obtained supernatant was isolated using automatic station QIAsymphony (Qiagen, USA). Concentrations of DNA were investigated using the TaqMan-PCR for a fragment of the gene of the household and GAPDH for genetic marker RH supplies RHD (9) (table 3).

As can be seen from table 3, the proposed method allows, on average, 6.5 times to increase the concentration of vjnk. The concentration of fetal DNA when using the proposed method increases by an average of 18%. This fact can be very important when Ana�ize fetal DNA in women with early pregnancy when the concentration of fetal DNA is very small. Using the proposed method of processing blood may allow RhD typing of the fetus earlier in the pregnancy.

Example 4

In groups of patients with lung cancer (n=10) and healthy donors (n=10) was investigated the level of expression in the blood circulating microRNAs (miRNAs) on the example of miRNA-21 and 155, the level of expression of which increases with the development of cancer pathology (10). Obtaining fractions vnrc conducted using the prototype method (6) and the proposed method. Everyone participating in the study was taken by the blood through the tube. The collected blood was divided into 2 equal parts, one part was processed in three stages according to the Protocol prototype, and the second part was treated in one stage analogously to example 1, with vnmc from the obtained supernatant was isolated using guanidine phenol extraction (11). The expression level of miRNAs targets were determined using quantitative RT-PCR (12). It was shown that using the proposed method of obtaining the total fraction vnrc blood allows to increase the output vnrc targets on average by 32%.

Using the proposed method allows to simplify and shorten the duration of the procedure and increase the number of diagnostic material that will improve the sensitivity of detection Marche�tion NC, specific to cancer and pathologies of pregnancy.

Sources of information

1. Jung K., Fleischhacker M Rabien A. Cell-free DNA in the blood as a solid tumor biomarker-a critical appraisal of the literature // Clin Chim Acta. - 2010. - V. 411. P. 1611-1624.

2. Pinzani P., Salvianti F., M. Pazzagli et al. Circulating nucleic acids in cancer and pregnancy. // Methods. - 2010. - V. 50(4). P. 302-307.

3. Rykova, E. Y., E. S. Morozkin, A. Ponomaryova A. et al. Cell-free and cell-bound circulating nucleic acid complexes and ecological feasibility study: mechanisms of generation, concentration and content // Expert Opin Biol Ther. - 2012. - V. 12. P. 141 to 153.

4. Skvortsova I.e., Rykova, E. Y., S. N. Tamkovich, et al. Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation // Br J Cancer. - 2006. - V. 94. P. 1492-1495.

5. Rykova E. Yu., Skvortsova T. E., Hoffman A-L. et al. / / Circulating extracellular DNA and RNA in blood in the diagnosis of breast cancer pathology. Biomedical chemistry. 2008, Volume 54, Issue 1, Pp. 94-103.

6. Laktionov P. P., Tamkovich S. N., Rykova E. Yu. a Method for early diagnosis and monitoring of cancer. Patent RU 2251696 C2, publ. 10.05.2005.

7. Morozkin, E. S., Babochkina T. I., Vlassov V. V., Laktionov P. P. The effect of protein transport inhibitors on the production of increasing interest among DNA // Ann N Y Acad Sci. - 2008. - V. 1137. - P. 31-35.

8. Ponomaryova A. A., E. Y. Rykova, Cherdyntseva N. V. et al. / RARβ2 gene methylation level in the circulating DNA from lung cancer patient blood // Eur J Cancer Prev. - 2011 V. 20, N. 6. - P. 453-455.

9. F. B. Clausen, T. R. Jakobsen, K. Rieneck et al. Pre-analytical conditions in non-invasive prenatal testing of cell-free fetal RHD // PLoS One. - 2013. - V. 8(10). e76990.

10. Jones K., Nourse, J. P., Keane, S. et al. Plasma microRNA are disease response biomarkers in classical Hodgkin's lymphoma // Clin Cancer Res. - 2014. - V. 20. - P. 253-64.

11. Chomczynski P., SacchiN. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction // Anal Biochem. - 1987. - V. 162. - P. 156-159.

12. Chen S., D. A. Ridzon, A. J. Broomer et al. Real-time quantification of microRNAs by stem-loop RT-PCR // Nucleic Acids Res. - 2005. - V. 33. - P. e179.

1. The method for allocating total fraction of extracellular nucleic acid from blood (VNC), including the collection of blood in antivertigo solution, separation of the collected blood into plasma and cellular fraction of blood by using centrifugation, separation VNC from the resulting supernatant with subsequent measurement of the concentration VNC, characterized in that the blood sample before separation into fractions, add an equal volume of elution buffer containing 1 M NaCl, 0.2 M NaHCO3(pH 9,3), 20 mm EDTA or buffer containing 1 M NaCl, 20 mm EDTA, the mixture was incubated at room temperature for 4-6 minutes with stirring.

2. A method according to claim 1, characterized in that VNC isolated from the supernatant by a method selected from: extraction with phenol-chloroform; extraction with guanidine-phenol method; isolation by ultra fine glass or glass-fiber filters in the presence chaotropic salts.

3. A method according to claim 1, characterized in that the measurement of the concentration VNC carried out by a method selected from: quantitative PCR and RT-PCR, methyl-specific�th quantitative PCR, method of measuring the concentration of TC using fluorescent dyes.



 

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