RU2521655C1 - Method of assessment of gene expression of tryptophanyl - total rna synthetase as marker of overwork during training of athletes - state of overtraining - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a method of determining in an athlete of state of fatigue and a state of "overtraining" on increased gene expression of tryptophanyl-total RNA synthetase (TRSase). The method comprises taking a control sample from the athlete prior to intense training and the test sample after an intense training. As the sample the sample of blood or scraping or flushing with the oral cavity is used. The obtained cells are selected and washed. Total RNA is isolated from the cells. Reverse transcription is carried out and then amplifying of the cDNA obtained. The gene expression in TRSase is assessed in test and the control samples. Fatigue state and the state of "overtraining" is determined in case of a significant increase in gene expression level of TRSase in the test sample compared with a control sample, namely more than 1.45 times.

EFFECT: invention enables to increase significantly informational content, to simplify and accelerate the procedure of testing.

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Description

The invention relates to biotechnology. The invention allows to significantly increase the information content, relatively simplify and speed up testing of the level of physical fitness and fitness of athletes and the degree of development of excessive fatigue and assess the risk of maladaptation with the development of overtraining syndrome and loss of physical fitness with a sharp decrease in immunity. At the same time, it is possible to search for new training techniques in tactical and strategic planning in sports.

Current level of research and knowledge

Despite a long period of studying the complex of enzymes of extra-ribosomal protein biosynthesis - aminoacyl-tRNA synthetases (APCase) remains incompletely understood. This class of enzymes, which arose at the dawn of the evolution of living organisms and the appearance of the first multicellular organisms, was “adapted” to perform a wide range of additional “noncanonical” regulatory functions. Since the most important function of the body is protective and regulatory, these functions were “devoted” to various forms of ARSaz, among these functions one can list the regulation of angiogenesis, active participation in the implementation of the complex of natural immunity reactions, autoimmune reactions, a whole range of activities to suppress the development of tumors, a complex anti-inflammatory reactions and inhibition of the development of bacterial pathogens [1]. ARSases, in order to optimize their basic functions to ensure the specific attachment of amino acids to the corresponding transport RNAs, form large multienzyme complexes in the cell, which include a number of cofactors with important regulatory functions. A number of ARSases are not included in such complexes, can be secreted into the bloodstream and have a spectrum of regulatory cytokine functions - in particular, tyrosyl-tRNA synthetase (TirRS) and TRS. Of particular interest in connection with the studied function of the marker of the physical state and the state of overtraining is TRSase. This enzyme is strongly induced by the antiproliferative cytokine interferon γ, is involved in suppressing the growth of pathogens, provides stimulation of the synthesis of antibodies - immunoglobulins, when modified by matrix metalloproteinases, it acquires a pronounced antiangiogenic function, inhibits the development of tumor metastasis, is involved in the synthesis of diadenosine polyphosphates, A 3 also combined with a number of factors anti-oncogenic effect [2]. Such a large set of additional regulatory functions of the enzyme makes it an important object for use as a diagnostic feature and as an object of corrective and therapeutic action.

Detailed studies of the dynamics of the activity of the immune system of athletes during intensive training showed that the behavior of the physiological parameters of the immune system differs depending on the intensity of training and the personality of the trained athlete. The state of overtraining among elite athletes is often accompanied by respiratory diseases (RH), which has a detrimental effect on performance during responsible competitions and has already led to the loss of a significant number of potentially possible gold medals in the past Olympic Games in a number of national teams. The behavior of the immune system in this case is described by the "J" form of dependence of the intensity of training on the probability of RH. At the same time, the state of the immune system is also described by the presence of an “open window” of impaired immunity with a high risk of RH, which lasts after intensive training for up to several days. It should be emphasized the great individual specificity of reactions to intense training and the achievement of a state of "overtraining", while it is especially important to have an informative and reliable marker of this state of the body. Due to the weakening of the system of specific immunity of blood cells, additional protective mechanisms are sharply activated in the body, in particular, the activation of non-canonical protective mechanisms of ARSaz. The cascade of anti-inflammatory and anti-proliferative reactions is triggered by interferon γ, one of the consequences of which is the powerful activation of secreted indolamine 2,3-deoxygenase (IDO), which catalyzes the first stages of the breakdown of the amino acid tryptophan, which leads to local depletion of the extracellular tissue environment by the essential amino acid tryptophan. In this case, competition with pathogenic bacteria for the amino acid tryptophan occurs. In parallel, in the cells of the corresponding tissues, including blood cells, the expression of TRSase is strongly stimulated, which binds intracellular tryptophan with the corresponding tRNA and “conserves" AA for the needs of the cells of the body. Another function of a sharp increase in the expression of TRSase in the blood cells responsible for immunity is the synthesis of immunoglobulins significantly enriched in tryptophan. Our choice of TRSase as a marker of the state of suppression of the immune system during overtraining is based not only on the properties already noted. A decrease in the functionality of the immune system significantly increases the risk of the onset and development of the oncological process. In the process of increased expression in the cell, TRSase is able to secrete into the intercellular space and blood, undergo activating modifications and significantly suppress the processes of pathological angiogenesis, thereby blocking the further development and metastasis of primary tumor cells [1]. Finally, TRSase is one of the important producers of Ap ₃ A diadenosine polyphosphate, which has a strong tumor inhibitory effect [3].

Known biochemical markers of overtraining - plasma glutamine.

Plasma concentration of glutamine has been proposed as a possible indicator of stress from overtraining, as this results in low levels of plasma glutamine in athletes with OT. Plasma concentration of glutamine decreases after intense or prolonged training, but not after intensive and short-term training. Glutamine reduction can occur after physical injuries, burns, inflammation, and infections. Plasma concentrations of glutamine temporarily increase after protein nutrition, but it decreases by 25% after several days of low carbohydrate intake (4).

Enzyme Assessment - Creatine Kinase Activity

Evaluation of the activity of the creatine kinase enzyme is widely used, and not quite as an OT marker, but as a tool for identifying recent muscle damage or POT (4). This fact explains that well-trained athletes performing eccentric muscle exercises do not show a significant increase in creatine kinase activity, even if they feel muscle pain, possibly due to the fact that they are the result of damage or inflammation in the connective muscle tissue (4). On the other hand, a diagnosis based on the definition of creatine kinase seems reasonable and adequately measures the increase in muscle tension or individual tolerance of muscle loads.

It has been suggested that the concentration of nitrogen residues in the blood plasma (urea and uric acid) may indicate a decrease in muscle protein and, as a result, be an OT marker due to an obvious connection with the catabolic state. The biggest problem is that intense and lengthy workouts associated with a temporary increase in urea and uric acid indices may depend on diets with protein intake. For these reasons, urea, uric acid, and creatine kinase do not represent reliable parameters for the unambiguous and accurate detection of RT.

Control of biochemical markers of the degree of fatigue - the level of urea and lactate in the blood.

A decrease in maximum physical endurance in parallel with a decrease in maximum concentration of lactate has been described for runners, swimmers, cyclists and triathletes (4). Decreased blood lactate in response to submaximal tests was observed in athletes with RT, probably due to a decrease in muscle glycogen index, a decrease in catecholamines in response to exercise, or a decrease in the effect of catecholamines on muscle tissue. Determining blood lactate is a training routine for athletes, so it is important to know if it can be used as a tool for diagnosing OT. In this context, it was proposed to study the physiological reactions to the endurance of athletes for 4 weeks with an increase in the volume and intensity of training. The lactate level was evaluated and it was concluded that athletes who entered the OT state with a significant increase in workload showed a reduced lactate level during the maximum load test. Thus, lactate can be an important tool for preventing OT, as it is an affordable and widely used method in the field of sports.

Assessment of hormonal status. Hormonal markers: test for the ratio of testosterone / cortisol. The balance between anabolic and catabolic activities is determined by the ratios of testosterone and cortisol, which is known as the testosterone / cortisol test or free testosterone / cortisol. Based on the fact that testosterone has an anabolic effect, a cortisol catabolic testosterone / cortisol coefficient has been proposed as an important marker of OT. It is suggested (4) that if the decrease in this indicator is more than 30%, then the athlete will be in a state of OT. In a series of experiments, this position was confirmed. Since the analysis was carried out by non-invasive tests in the athlete's saliva, the method was widely used.

Disclosure of invention

Analysis of the expression of TRSase gene mRNA genes in blood samples and scrapes of the epithelium of the oral cavity allows one to obtain reliable information about the deep molecular processes that accompany muscle contractions of various shapes and intensities and to accurately register stress states in muscles with the risk of maladaptation processes in muscle tissues and other organs and tissues individual, dramatically reducing his physical parameters and health and, most importantly, or register the beginning of a destructive destructive process s, accompanied by a sharp drop in immunity.

The implementation of the invention

The technical result achieved by the implementation of the present invention is a significant increase in the information content, efficiency and speed up testing of the physical condition and level of the dangerous state of overtraining, as well as the possibility of prompt correction of training methods. The procedure for testing the state of overtraining includes taking a control sample of blood or scraping or rinsing from the oral cavity from an athlete before intensive training and a test sample after intensive training, selecting and washing the obtained cells, isolating total RNA from them, performing reverse transcription, amplification of the obtained cDNA, assessment expression of the TRSase gene in the experimental and control samples, while the state of fatigue and the state of "overtraining" is determined in the case of a significant increase in the level of I TRSazy gene expression in the test sample compared to a control sample (more than 1.45 times). One of the essential aspects of the invention is the possibility of non-invasive testing.

Definition of Terms

The level of physical condition is understood to be measured by the classical physical (step test, Cooper test, etc.) and physiological methods (VO ₂ max) the degree of physical form or fitness of the test subject [4].

The state of “overtraining” means a complex of responses of the body to intensive training, exceeding the body’s ability to normal recovery process, including sharp changes in metabolism and a complex of immunological protective reactions, expressed in a significant change in the level of neurohormones and their receptors in tissues and a decrease in the sensitivity of muscle tissue to catecholamines, as well as a sharp increase in the level of leukocytes and inflammatory cytokines in the blood, which leads to a significant a decrease in resistance to infection and the emergence of the so-called "open window" for infections.

The DNA and RNA preparations obtained from the method of magnetic nanoparticles from blood are understood as the method of using specially treated particles of silica gel and their use to isolate pure and non-degraded DNA and RNA preparations [3].

A non-invasive test refers to the taking of biological samples by scraping, special sterile scrapers, or ear cotton swabs with transfer to a sterile isotonic saline solution.

The procedure for determining gene activity by expression of the TRSase gene mRNA means the procedures for obtaining RNA preparations from biological samples, transferring it under sterile conditions to cDNA, followed by recording the level of specific mRNA (cDNA) by RT PCR using fluorescently labeled or biotinylated probes, or using fluorescent dyes and calibration curves, with accurate identification of the specificity of the resulting amplification.

A comparative analysis of the proposed method for assessing the state of "overtraining" with similar patents-analogues.

To compare the proposed method with existing analogues (proposed by FIPS specialists of the Russian Federation) (patents No. 891095, 2056860 C1 and 2429836 (13) C1), it is necessary to note the complexity of the problem and its relevance, due to the increased susceptibility to sharp drops in the activity of the immune system and resistance to infections, it is in the case of elite athletes.

The system of natural nonspecific immunity responds to the chronic stress of intense training with a sharp increase in the activity of NK-killer cells and suppression of the phagocytic anti-infection function of neutrophils. Despite the obvious connection between intensive training and the functions of neutrophils, there is evidence that free radical forms of oxygen (ROS) produced by neutrophils are important modulators of muscle activity, antioxidant defense, and recovery in the physiological response. A number of important mitochondrial biogenesis factors, such as the PGC-1 alpha coactivator, AMPK kinase, and SIRT1, are activated in response to changes in the redox potential of cells caused by intense exercise. Activation of PGC-1 alpha leads to a decrease in the damaging effect of ROS by stimulation of antioxidant enzymes and / or an increase in the number of mitochondria, which makes it possible to produce the same amount of ATP at a lower level of ROS. Moreover, ROS compounds induced by intense training can be important for DNA methylation and post-translational modification of histones, which allows reaching the state of inherited adaptive states based on epigenetic modification of chromosomes [Radak Z., Zhao Z., Koltai E., Ohno H. , Atalay M. Oxygen Consumption and Usage During Physical Exercise: The Balance Between Oxidative Stress. and ROS-Dependent Adaptive Signaling Antioxidants & redox signaling, 2013, v.18 (10), p.1208-1246]. Thus, changes in the phagocytic activity of neutrophils registered in the analogues proposed (patents Nos. 891095 and 2056860 C1) are most likely normal physiological reactions that are not directly related to the state of “overtraining”. A very different reaction to intensive training of neutrophil functions in different individuals also reduces the diagnostic value of the criterion used, making it uninformative.

One of the drawbacks of the analysis of many parameters of the immune system is their high cost and focus on only one aspect of the most complex set of responses to intense training, which have many complementary and interchangeable levels of adaptation. A number of functions of the immune system are affected by both intense and moderate prolonged training, tissue damage and infection. In addition, the technical tools used in the analogue patent No. 2056860 C1, to enhance the diagnostic value of the phagocytic activity of neutrophils, which include the use of piezoelectric sensors, radio transmitters and receiving remote devices, allow to record the pulse and technical parameters of the athlete moving along the track and the state of overwork, which is also not It is directly related to the state of "overtraining".

Comparison with patent analogue No. 2429836 (13) C1, only confirms the validity of the proposed criteria for assessing the state of fitness and overtraining. Understanding the multifactor, multicomponent, and complementarity of the body’s responses to intensive training, in which the decrease in the protective effect of blood cells (neutrophils, monocytes, lymphocytes, etc.) that carry out phagocytosis, lysis of infectious agents, foreign cells and cell debris, is simultaneously triggered, is crucial. natural immunity reactions that carry out immune suppression reactions, the important link of which is the expression of TRSase, moreover, as the responses to stress associated with intense training go along almost the same mechanism as the response to an infectious effect, the expression level of this gene is a criterion of the strength of the response, strictly adequate to the strength of the stress, and exceeding a certain “threshold” value of stress from physical activity during training and starts a complex of "adaptive" reactions of the body, expressed in the reaction of the syndrome of "overtraining". The use of the drug trekrezan to activate the protective reactions of the body, apparently, acts in a complex manner, while simultaneously activating proliferative defense reactions, stimulating both specific and nonspecific immunity, including through the activation of TRPCase expression and related antiangiogenesis and anti-oncogenesis reactions [Yao P., Fox pl Aminoacyl-tRNA synthetases in medicine and disease. EMBO Mol Med. 2013, v.5 (3), p.332-343].

Examples of own experiments

Our own studies have established that determining the level of expression of TRSase mRNA (RF patents RU No. 2429836 C1 dated 04/01/2010 “Method for monitoring the physiological state of a person”) can also be used to monitor the physical condition of an individual at various stages of the training process and achieve a dangerous state of overtraining with the purpose of the necessary process adjustments and increase the efficiency of adaptation processes and increase the athlete's physical form. The relevance of the cycle of methodological experiments given below and revealing the essence of the independent attribute (item 1) is associated with the practical lack of a reliable method for diagnosing the state of overtraining at present, as well as the great potential for improving the methodology, both in terms of accurate diagnosis of the state of overtraining and the possibility of correcting the state of the body, both in the case of healthy individuals, and possible pathologies.

Our own studies have shown that the level of TRSase gene mRNA expression is a highly effective criterion for the general condition of various systems of an athlete’s organs and allows developing optimal training tactics.

It should be emphasized the interconnection and interdependence of the following methods, their relationship with independent features (item 1) from the point of view of disclosing the content of these features. A preliminary assessment of the specificity and level of expression of TRSase mRNA was determined by a set of methods described in Example 1, which consisted of scanning gels with a picture of separation of specific PCR cDNA products for the content of copies of TRSase mRNA, in parallel with markers of DNA quantities for constructing a calibration curve. The final assessment of the expression level selected at this stage of the analysis, used as a diagnostic feature, was already used by real-time PCR using the constructed calibration curve (example 2) or using the method of relative quantitative assessment of mRNA using the reference gene (example 3). A brief description of the advantages and disadvantages of the techniques in terms of the need for the full implementation of the provisions of the independent features of the invention is given (1).

The following are examples illustrating the invention.

Example 1

We have developed a set of methods for detecting the state of fatigue by determining the level of activity of the gene encoding TRSase by the specific mRNA synthesized from the gene. The sequence of operations of the developed methods included the selection of scrapings from the oral cavity, the selection and washing of the obtained cells, obtaining microquantities of blood from a finger, isolation of RNA and DNA preparations using Silex nanoparticle kits. RNA was also isolated using the Silex Yellow Solve kit. RNA was converted to complementary DNA using reverse RNA-dependent DNA polymerase (MMoLV) using degenerate hexanucleotide seeds. The formed specific TRSase cDNA was detected using specific PCR and specially selected seeds that propagated exactly the desired types of cDNA, rather than possible impurities of genomic nuclear DNA. The gels were stained with ethidium bromide or Siber-Green dye.

Quantitative analysis of specific tryptophanyl tRNA synthetase mRNA.

Expression of the TRSase gene was quantified by the level of specific mRNA. In this case, a quantitative analysis of the total cDNA determined by PCR with subsequent analysis of the obtained electrophoregram densitometrically by the brightness of the luminescence of the products served as a criterion for the mRNA level. During the PCR reaction, Silex-M reagent kits were used (http://sileks.com/ru/), specific seeds were synthesized at Syntol. The seeds were designed according to the program recommended by the manufacturer.

Isolation of RNA preparations. After taking control and experimental samples and washing the cells (in accordance with paragraph 1), take about 100 μl of a suspension of epithelial cells after taking scrapings from the oral cavity or blood sample in 0.05 M EDTA, add 1 ml of YellowSolve and mix. The lysate is centrifuged at 10-15 thousand rpm for 5 minutes Add 0.1 ml of chloroform to the supernatant, vortex the mixture vigorously, and incubate for 20 minutes at room temperature by shaking the tube every 5 minutes. Centrifuge (CF) the tube at 10-15 thousand rpm./min for 5 minutes The upper layer is selected, the lower layer is discarded. Add the equal volume of GreenClean deproteinizing solution to the selected top layer and vigorously shake the mixture on a vortex for 2-3 minutes, CF 10-15 thousand rpm for 5 minutes. 2 volumes of 96% ethanol are added to the supernatant, incubated at -20 ° C for 20-30 minutes to form an RNA pellet. Precipitate CF RNA at 10-15 thousand rpm./min for 10-20 minutes The precipitate is washed with 0.5 ml of 80% ethanol and centrifuged at 10-15 thousand rpm./min for 15 minutes Dissolve the RNA pellet in bidistilled sterile water and freeze at -20 ° C.

Reverse transcription. The following components are mixed in a test tube placed on ice: RNA solution 2 μl (0.1-5 μg) or poly (A) plus RNA 10-0.5 ng, seed - 1 μl 0.5 μg hexaprimer “random” (or oligo (dT ) 15 0.5 μg) RNase-free water up to 18 μl. Mix, precipitate drops by short-term centrifugation. Incubate the mixture for 5 min at + 70 ° C, transfer the tube to ice and collect droplets by short-term centrifugation. Then the following components are added: 10-fold RT buffer 2.5 μl, 4 μl 2.5 mm dNTP mixture, M-MLV reverse transcriptase 0.5 μl (100 units act). The mixture is then incubated for 60 min at + 37 ° C (if using random hexaprimers, before incubating at + 37 ° C, incubate the mixture for 10 min at + 25 ° C and then incubate the mixture for 60 min at + 37 ° C). Stop the reaction by warming the mixture for 10 min at + 70 ° C. Transfer the mixture to ice. The resulting cDNA can be directly used immediately for PCR or second strand synthesis and subsequent cloning. Store synthesized cDNA at -20 ° C or at -70 ° C for long-term storage.

Polymerase chain reaction. The polymerase chain reaction is carried out in a volume of 50 μl of the reaction mixture containing 80 pmol of each of the two primers (1 μl of a solution in 1 st / ml), 5 μl of a 10 mM dNTP solution, 5 μl of 10 buffers (100 mM Tris pH 8, 5 at 25 ° C, 500 mM KCl, 1% Triton X-100), and 2 units of Taq DNA polymerase. Mixtures for a series of PCR reactions are mixed as a total “master mix”, which is then divided into the planned reactions, to which 1 μl of cDNA from control and experimental samples was added (the baseline resting state of an individual or after intense training). PCR was carried out in the following mode - 94 ° C for 15 s, from 58 ° C to 62 ° C for 15 s and 72 ° C for 30 s. Aliquots of 7 μl of reaction were selected to control the progress of the reaction.

The seeds used to obtain a specific PCR fragment of TRSase mRNA from total cDNA copies of total RNA from biological samples:

- straight = 5'-GAAAGGCATTTTCGGCTTCA-3 '

- reverse = 5'-CAGCCTGGATGGCAGGAA-3 '.

In the series, all reactions were repeated twice. Standard samples for comparison were obtained by amplification of control samples of the biological material of athletes before intensive training. Relative expression values were estimated by densitometric analysis of PCR products of analysis of RNA samples, after their conversion to cDNA and amplification using specific seeds as described below. As control values taken as 100%, samples of biological material before training began.

In accordance with clause 1 that “the state of“ overtraining ”is determined in the case of a significant (more than 1.45-fold) increase in the level of expression in the experimental sample compared to the control", the main diagnostic sign is precisely the level of TRPCase expression. And this follows the great importance of accuracy, reproducibility and mutual complementarity of the above methods for the quantitative evaluation of copies of TRSase mRNA in RNA and cDNA preparations.

The presented methods reflect the current strategies for the quantitative assessment of the formed specific types of cDNA, reflecting the initial content of specific mRNA of TRSase.

An adapted and optimized method for the quantitative assessment of specific types of cDNA (types of TRSase mRNA) by gel electrophoresis.

A comparative analysis of the advantages and disadvantages of this approach in the “hierarchy” of the above methods allows you to position it as dependent, allowing you to isolate a group of subjects with a high risk of “overtraining”.

Electrophoresis was performed on a 7% polyacrylamide gel in Tris-borate buffer pH 8.3 (89 mM Tris, 8.9 mM boric acid, 2 mM EDTA). Aliquots from the reaction mixture were applied to the pockets of the gel in a volume of 7-10 μl. Electrophoresis was carried out at a voltage of 10 V / cm for 1.5-2.0 hours. The gel was stained with a solution of ethidium bromide 1 μg / ml for 15 minutes Images of gel electrophoresis were photographed using a set of equipment, including a UVT-1 transilluminator, a Kodak DC120 digital camera, a system for monitoring gels and registering gels, ViTran Photo, and an image processing system, ViTran Photo. Digital copies of the gels were analyzed using Eastman Kodak's EDAS 120 Electrophoresis Documentation and Analysis System 120 software package. Kodak Digital Science 1D Version 2.0.3 was used to measure band intensities. A quantitative assessment of the content of specific types of cDNA (and the corresponding content of TRSase mRNA types) in the samples deposited on the gel was calculated by the intensity of the DNA bands of the molecular weight marker, the exact concentration corresponding to the mobility in the gel.

Figure 1 shows the results of a series of studies of the relative expression of the TRASase gene of athletes of the Russian men's water polo team. The values of relative expression were estimated by densitometric analysis of the pattern of electrophoretic separation of PCR products of amplification of samples of total RNA preparations, after their conversion to cDNA and amplification using specific seeds. As control values taken as 100%, samples of biological material before training began. The data presented are averaged data from 2 parallel experiments. In the following examples, individual samples from this panel were investigated using alternative approaches including RT PCR and absolute and relative methods for estimating the amount of specific mRNA of the TRCase gene in biological samples.

It can be seen that in samples No. 2, 9, 12, 13, expression intensifications are going on, significantly exceeding the base level, indicating activation of recovery processes that occur in response to excessive loads and requiring the use of individual recovery programs.

Comparative characteristic of the method. The advantage of the method is its relative simplicity, less stringent requirements for the specificity of PCR seeds, relative cheapness due to the non-use of fluorescent probes and expensive equipment for real-time PCR (the cost of an RT-PCR device is from 1.5 to 5 million rubles). The main disadvantages are the low accuracy of the methodology, the complexity of mass analysis (hundreds of samples per day) in connection with the procedures of electrophoresis and gel scanning. However, it allows isolating the “risk group” of athletes for further more accurate analysis, which reduces the cost of the overall testing procedure (especially if it is necessary to analyze several gene biomarkers).

Example 2

Comparative characteristic of the method. The given example is a further methodological development of methods for quantitative assessment of nucleic acid populations in mixtures in general, and as applied to the assessment of "overtraining" in particular. The method allows you to record the level of expression of specific mRNAs "in control and experimental samples" in real time, which allows you to make a final conclusion about the state of training. The method contains all the main steps and procedures of the previous methods reflected in the claims, including “collecting samples, washing cells, isolating RNA, obtaining cDNA” and performing specific real-time PCR with determination of the absolute amounts of cDNA copies of transcripts of mRNA TRSases. This method does not use expensive fluorescent probes.

Analysis procedures. There are two approaches to quantifying gene expression by real-time PCR (RT PCR): relative and absolute quantification of gene expression. The relative method for quantitative comparison of gene expression is based on comparing the level of gene expression in one sample with the expression of another standard reflex gene used for normalization. Absolute quantification is based on the use of a standard calibration curve, which is obtained from matrix samples with a precisely known concentration. The concentration of any unknown DNA or RNA preparation can be determined by simply comparing its PCR signal (Cq) with a standard curve.

The sequence of procedures for the quantitative determination of the expression level of the TRSase gene (or another gene) using the absolute quantitative approach in the RT PCR system includes: RNA extraction, cDNA synthesis, preparation of serial serial dilutions, RT PCR amplification and data analysis.

1. RNA extraction and evaluation of the quantity and quality of the drug.

A feature of the approach used in this example is the mandatory requirement for high quality and integrity of RNA preparations. For this, it is necessary to observe increased precautions (maximum speed of collection and storage of samples in special solutions) when collecting and transporting samples, as well as when isolating and storing total RNA preparations. The quality and quantity of the obtained preparations of total RNA was checked by UV spectroscopy.

To save the resulting preparations of total RNA, the samples were diluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and its optical density was measured at 260, 280, and 320 nm. To take into account the background absorption during light scattering caused by contamination of the cuvette and dust particles, the optical density data at 320 nm are subtracted from the data at 260 and 280 nm. The purity of the obtained RNA preparations was determined by the ratio of A260 / A280. Values between 1.8 and 2.1 are indicative of a high degree of purification of RNA preparations. The concentration of the RNA preparation was estimated by the formula: RNA concentration (μg / μl) = (A260 × 40 × D) / 1000, where D is the dilution coefficient. For example, we diluted 2 μl of the RNA preparation in 498 μl of TE, pH 8.0, to obtain A ₂₆₀ = 1.0, then the concentration of the RNA preparation was 10 μg / μl. RNA preparations after isolation during further analysis procedures were stored in ice. For long-term storage, the drug was frozen at -80 ° C, but it is preferable to immediately transfer it to cDNA form.

2. Synthesis of cDNA. The synthesis of cDNA was carried out mainly as in the previous example. Specificity lies in the increased requirements for the quality of the drug and the requirement of completeness and thoroughness of the selection of reaction conditions. For the reaction, from 0.1 pcg (picograms) to 1 μg of the total RNA preparation (spRNA) was taken. In the final volume of the reaction mixture, 10–20 μl combined up to 5 μg of spRNA, 1 μl of seeds (50 ng / μl of hexanucleotide seeds), buffer for annealing of seeds (1-2 μl) and ddH ₂ O (bidistilled water) to the corresponding volume of the total mixture . Incubated for 5 min at 65 ° C and immediately placed in ice for 1 min. Short centrifugation (CF).

Then add 2X buffers for the synthesis of the first cDNA strand (10 mM MgCl2, 1 mM each dNTP) and 2 μl reverse transcriptase, mix briefly with CF and incubate for 10 minutes at 25 ° C, then 50 minutes at 50 ° C. Stop the reaction by incubation at 85 ° C for 5 minutes. Quickly cooled in ice. Immediately proceeded to the next stage. Otherwise, cDNA preparations were stored at -20 ° C.

3. Preparation of serial serial dilutions of the obtained cDNA preparations. In order to ensure thorough and uniform coverage of the required quantitative range of cDNA, a sufficient degree of dilution of the samples should be prepared to cover the expected range of gene expression in the studied samples. We have prepared at least 5 points of 10-fold serial dilutions for a standard curve that can be used by RT-PCR software to determine the concentration of obtained cDNA prototypes. To construct the calibration curve, 18 μl of non-nucleus water was added to 4 tubes, briefly CF and labeled with numbers 2 through 5. The cDNA preparation obtained earlier was thawed (tube 1), mixed well and transferred 2 μl to tube 2. It was mixed by pipetting. And so on until test tube 5.

4. Carrying out the RT PCR procedure. Real-time standard PCR takes from 30 to 120 minutes. The protocol used is applicable to standard and fast real-time PCR. To perform specific PCR, in accordance with the example given, the following protocol was used: 1. Add 2 μl from each dilution and separately 2 μl of water (control minus matrix, or -MM), in duplicate in a 48-well plate; 2. When using unknown samples, add 2 μl each, preferably in duplicate, to the remaining free wells on the plate. In this case, the software will automatically use the standard curve (wells from A1 to E2 in the experimental scheme) to determine the concentration of unknown samples; 3. Prepare RT PCR "master mix" (6 μl of non-nucleated water, 10 μl of 2X "master mix", 2 μl of 10X seed to a concentration of 100-900 nM) in a volume of 10-20% more than necessary to account for dosing errors when the reaction mixtures are spaced in the wells of the plate; 4. Add 18 μl of the master mix to all wells containing dilutions, control samples, or test samples. Gently mix by pipetting, avoiding bubbles; 5. Seal the plate, briefly CF, and place in the RT PCR device; 6. Select (enter) a suitable (pre-optimized) program for the experiment from the proposed protocols; 7. In the window for setting the parameters of the instrument program indicate the wells of 10-fold serial dilutions (A1-E2), as previously described in the experimental design, indicate the controls and experimental wells (A3-F8) of the plate; 8. They start the device and during the process you can visualize the amplification in real time in the Run Monitor window. Amplification takes about 40 minutes (fast protocol) or up to 90 minutes (standard protocol).

5. Data analysis. After completing the procedure, the program automatically opens the “data analysis” window and performs basic analysis procedures, automatically determining the baseline and threshold values. Next, select the function "amplification schedule". To extract quantitative data from real-time PCR amplification curves, the results should be plotted as a linear regression of the Cq values versus the logarithm of the RNA / DNA ratios (i.e. the standard calibration curve). Instrument software automatically generates a standard curve. To view the curve, select the “results” tab in the “data analysis” window.

PCR data extracted from standard curves. The slope of the curve of the graph is a measure of the effectiveness of PCR analysis. Slope values between -3.1 and -3.6 are considered acceptable (90% and 110% efficiency, respectively), while a slope of -3.32 indicates 100% efficiency. R2 is a measure of analysis performance and is a correlation coefficient between experimental results and data expected under ideal conditions. R2 values must be greater than 0.99. This means that> 99% of the total variation in the DNA samples that make up the calibration curve can be explained by the relationship between the obtained Cq values and their corresponding DNA concentrations. To extract quantitative data from any Cq value, the following equation is used: Quantity = 10 ^{(Cq-b) / m} , where b is the y-intersection on the y axis, am is the slope of the linear regression.

Quantitative data are displayed either as weighted amounts of the original specific matrix (for example, picograms), its concentration (for example, picograms / μl), or as “copies” of the gene being studied. In the approach used for absolute quantitative PCR experiments, the units of the experimental samples should correspond to the units used in constructing the calibration curve. For more information, visit: http://www.illumina.com/support.

Figure 2 shows the result of a final series of experiments (using parallel biological samples in the experiments described in Figure 1 in Example 1 based on the absolute quantitative approach described in Example 2 to determine the amounts of expressed TRSase mRNA by constructing a calibration curve.

Comparative analysis of real-time PCR methods. The method requires high specificity of seeds, since the total amount of amplified material is estimated, including possible non-specific copying, self-copying of seeds, etc. The method is sensitive to copying errors, the need to construct calibration curves complicates the procedure, and requires many additional PCR reactions in each experiment. The advantage is the high accuracy of the method and obtaining data on the absolute amounts of cDNA species, which in some cases is important.

Example 3

Comparative characteristic of the method. The method for assessing the relative amounts of mRNA species back transcribed into cDNA is the most effective accurate and fast method. The use of fluorescence probes further increases the specificity of the technique, allowing only certain propagated types of cDNA to be detected that hybridize with a probe that allows the main independent trait (1) to be realized - more than 1.45-fold excess of the TRCase mRNA expression level. The main disadvantage of the method is the relative high cost of the reagents. The method is practically indispensable when it is necessary to assess the relative expression of several genes in one sample.

The relative quantitative method is based on the use of a “reference” gene, that is, a gene whose expression is constant in the test samples. The main advantage of the methods used in this example is the ability to mitigate errors in the deposition of reagents into the reaction mixture (mainly RNA / DNA matrices). To determine the relative expression of the studied gene in the sample using the normalizer gene, it is required to establish the expression level of both genes using RT PCR. For example, four C _T values should be set as indicated in table 1.

.Then we used the widely known method $$2^{-\Delta \Delta C_T}$$

.

метод. $$2^{-\Delta \Delta C_T}$$

method.

The method assumes that both genes (experimental and reference) have an amplification efficiency of about 100% (Hallmarks of an Optimized qPCR Assay). Then carry out the following manipulations:

1. Normalize the values of C _{T of the} studied gene with the values of the reference (ref) gene, both for the studied sample and for the calibration one:

∆C _{T (test)} = C _{T (research, test)} -C _{T (ref, test)}

∆C _{T (cal)} = C _{T (research, cal)} -C _{T (ref, cal)}

2. Normalize ∆C _{T of the} test sample with ∆C _{T of the} calibrator:

∆∆C _T = ∆C _{T (test)} -∆C _{T (cal)}

3. Assessed the expression level by the formula:

2 ^-∆∆CT = Normalized Degree of Expression

The result is the ratio of the studied gene in the sample with the value in the calibrator sample normalized by the expression of the reference gene. The process of normalizing the expression of the test gene with the reference gene compensates for any differences in the quantities taken for analysis of samples of biological material.

Calculation example: cDNA in the amount of 50 ng from RNA isolated from two compared samples (sick and healthy, trained and untrained) was analyzed by the studied gene and actin gene (reference gene). C _T values for each sample are shown below:

∆C _{T (normal)} = 15.0-16.5 = -1.5

∆C _{T (patol)} = 12.0-15.9 = -3.9

Then ∆C _{T of the} test sample is normalized with ∆C _{T of the} calibrator:

∆∆C _T = ∆C _{T (patol)} -∆C _{T (normal)}

= -3.9 - (- 1.5) = - 2.4

The final calculation is as follows:

$$2^{-\Delta \Delta C_T}=2^{-\left(-2.4\right)}=5.3$$

Conclusion: The studied samples have a 5.3-fold higher expression than in the control.

Direct work on this example included.

Express isolation of total RNA preparations (carried out as in example 1). To isolate total RNA preparations from non-invasively obtained samples of scrapings and / or swabs and blood samples, the Sileks-M Yellow Solve kit was used. The isolation procedure was carried out according to the protocol of the company, taking into account the specifics of the experiment. The RNA preparation was stored in alcohol at minus 20 ° C or lower.

Synthesis of cDNA RNA template. For the synthesis of cDNA from the resulting preparations of total RNA, reagents from Silex M and the attached protocols were used (see example No. 1). The obtained cDNA was used for RT PCR or for the synthesis of the second chain. The resulting cDNA was stored at -20 ° C.

Quantitative analysis of specific tryptophanyl tRNA synthetase mRNA.

Expression of the TRSase gene was quantified by the level of specific mRNA. In this case, a quantitative analysis of the total cDNA determined by RT-PCR was used as a criterion for the mRNA level. When carrying out the RT PCR reaction, Syntol reagent kits were used (R-402 reagent kit) in accordance with the company’s words. The level of specific cDNA for TRSase was quantitatively determined on an RT-PCR device. The seeds were designed using the Primer Express Version 2.0 program, with a check of the degree of specificity for the desired sequences using the Nucleotide BLAST database:

for β-actin:

- straight = 5'-CTGGAACGGTGAAGGTGACA-3 '

- reverse = 5'-CGGCCACATTGTGAACTTTG-3 ';

for cDNA copies of TRSase mRNA:

- straight = 5'-GAAAGGCATTTTCGGCTTCA-3 '

- reverse = 5'-CAGCCTGGATGGCAGGAA-3 '.

In the series, all reactions were repeated three times. For analysis of the results, a software package (RealTime_PCR v 7.3) was used, which was attached to the device. The level of deviations was calculated taking into account optimization. Standards obtained by amplification of control samples in a PCR reaction under conditions optimized for RT-PCR. The measurements were based on standard values obtained by 4-fold sequential dilutions and envelopes of the standard curve (8 points).

The results presented in Fig. 3 are a comparative analysis of the samples obtained before a series of trainings and after. Data for comparison was the relationship between the studied gene target of TRSase and β-actin mRNA. The figure indicates that when comparing the expression in individuals “before” and “after” intensive training, in a number of cases the expression of TRSase mRNA significantly increases - by 1.2-1.6 times. As in the examples using alternative methodological approaches, it can be seen that in samples No. 2, 9, 12, 13, expression intensifications are significantly higher than the baseline, indicating activation of recovery processes that occur in response to excessive loads and requiring the use of individual recovery programs.

Increased expression reflects the function of the enzyme in maintaining tryptophan levels for the synthesis of immunocompetent proteins rich in tryptophan residues. With the correct regimen of the training cycle, there is a decrease in the expression of TRSase mRNA, which is consistent with data on a decrease in the expression level of the cytokine complex, which are formed in response to the action of various stimulators of nonspecific immunity [11].

Literature

1. Guo M., Schimmel P., Xiang-Lei Yang X-L. Functional expansion of human tRNA synthetases achieved by structural inventions. 2010, FEBS Letters, V.584, N.2, p. 344-442.

2. Merkulova T., Kovaleva G., Kisselev L. P1, p3-bis (5'-adenosyl) triphosphate (Ap3A) as a substrate and a product of mammalian tryptophanyl-tRNA synthetase. 1994, FEBS Letters, V.350, p. 287-290.

3. Park S.G., Schimmel P., Kim S. Aminoacyl tRNA synthetases and their connections to disease. PNAS, 2008, V.105, N.32, p. 11043-11049.

4. Gleeson M. Biochemical and immunological markers of overtraining. Journal of Sports Science and Medicine, 2002, V.2, p.31-41.

Table 1 Test Calibration (Cal) Gene under study C _{T (research, test)} C _{T (research, feces)} Reference gene C _{T (ref, test)} C _{T (ref, cal)}

Claims (4)

1. A method for determining the athlete’s state of fatigue and the state of “overtraining” by increased expression of the tryptophanyl-tRNA synthetase (TRSase) gene, which involves taking a control sample from an athlete before an intensive training session and a test sample after an intensive training session, where a blood sample or scraping or rinsing from the oral cavity, selection and washing of the obtained cells, isolation of total RNA from them, reverse transcription, amplification of the obtained cDNA, evaluation of expression of the TRSase gene in op test and control samples, while the state of fatigue and the state of “overtraining” are determined in the case of a significant increase in the level of expression of the TRCase gene in the experimental sample compared to the control sample, namely more than 1.45 times. 2. The method according to claim 1, where the specific expression was evaluated densitometrically according to the electrophoresis of PCR fragments with marker bands of DNA. 3. The method according to claim 1, where the specific expression of TRSase mRNA was evaluated by the method of absolute quantitative assessment of mRNA according to RT PCR analysis using Sibrin Green dye and constructing a calibration curve. 4. The method according to claim 1, where the specific expression of TRSase mRNA was evaluated by the method of relative quantitative assessment of mRNA according to RT-PCR analysis using the method of determining the values of 2 ^-ΔΔCT using the "reference" β-actin gene.

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