RU2469323C2 - Diagnostic technique for bladder cancer (versions) and kit for implementation thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions involves a diagnostic technique for bladder cancer based on measuring the iRNA content of TRIB1 gene by PCR and a kit for implementation thereof. The higher iRNA content of TRIB1 in a provisionally involved bladder and/or urine as compared to its content in healthy tissue and/or blood is used as a diagnostic character of bladder cancer.

EFFECT: group of invention enables high-reliability diagnosing of bladder cancer, including at the early stage of progressing neoplastic aberration.

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Description

The invention relates to medicine, in particular oncology and molecular biology, and relates to a method for the diagnosis of bladder cancer (RMP) and a kit for its implementation.

Bladder cancer (RMP) is a common pathology. About 356,000 new cases of RMP are diagnosed annually in the world [Ploeg, M., K.K. Aben and L. A. Kiemeney, The present and future burden of urinary bladder cancer in the world. World J Urol, 2009.27 (3): p. 289-93]. Existing methods for diagnosing RMP are divided into two groups: invasive and non-invasive. Invasive diagnostic methods include cystoscopy, which allows visualization of the tumor and biopsy of the bladder, as well as transurethral ultrasound (transurethral ultrasonography) [Qu, X., X. Huang, L. Wu, et al., Comparison of virtual cystoscopy and ultrasonography for bladder cancer detection: A meta-analysis. Eur J Radiol, 2010]. All invasive methods are associated with discomfort and risk to the patient’s health, high cost and complexity of execution.

Non-invasive include the detection of RMP markers in physiological body fluids, transabdominal ultrasound tomography, X-ray computed tomography, magnetic resonance imaging, cytological examination of urine or wash fluid [Kenney, DM, RDGeschwindt, MRKary, et al., Detection of newly diagnosed bladder cancer, bladder cancer recurrence and bladder cancer in patients with hematuria using quantitative rt-PCR of urinary survivin. Tumour Biol, 2007.28 (2): p. 57-62, Van Tilborg, A.A., C.H. Bangma and E.C. Zwarthoff, Bladder cancer biomarkers and their role in surveillance and screening. Int J Urol, 2009.16 (1): p.23-30].

RMP biomarkers are divided into the following groups:

1) Markers, which are RNA genes differentially expressed in normal and tumor tissues.

2) Protein markers, as well as peptides and products of protein degradation, specifically detected in the urine or blood of the patient.

Various methods are used to evaluate the content of RNA markers of RMP, primarily microarray hybridization and real-time PCR (PCR-RV) [Livak KJ, Flood SJ, Marmaro J., Giusti W., Deets K. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4, 357-362].

As RNA markers of RMP use:

- survivin (elevated mRNA levels are found in tumor tissues, urine, and lavage fluid) [Kenney, DM, RDGeschwindt, MRKary, et al., Detection of newly diagnosed bladder cancer, bladder cancer recurrence and bladder cancer in patients with hematuria using quantitative rt-PCR of urinary survivin. Tumour Biol, 2007. 28 (2): p. 57-62];

- cytokeratin 20 (elevated levels of mRNA in urine and wash fluid) [Guo, B., C. Luo, C. Xun, et al., Quantitative detection of cytokeratin 20 mRNA in urine samples as diagnostic tools for bladder cancer by real-time PCR Exp Oncol, 2009. 31 (1): p. 43-7];

- hyaluronidase (mRNA level is elevated in the urine) [Van Tilborg, A.A., C.H. Bangma and E.C. Zwarthoff, Bladder cancer biomarkers and their role in surveillance and screening. Int J Urol, 2009.16 (1): p.23-30];

- telomerase (mRNA level is elevated in urine) [Eissa, S., M. Wellam, R. Ali-Labib, et al. Detection of telomerase in urine by 3 methods: evaluation of diagnostic accuracy for bladder cancer. J Urol, 2007. 178 (3 Pt 1): p.1068-72].

However, a significant drawback of all existing RNA markers of RMP is their low prognostic value (less than 20% of cases of RMP), which leads to poor reproducibility of the results and low clinical value of such tests.

For the diagnosis of RMP using protein markers, immunochemical analysis methods based on the reaction of interaction of specific antibodies with a protein marker are used. Using these methods, one can evaluate the quantitative content of the marker protein RMP and its spatial distribution in the tissue under study. The biological sample for analysis of the protein can be immobilized on a solid carrier, for example on a polymer membrane, on which cells, parts of cells or proteins can be immobilized. This carrier is subsequently hybridized with labeled antibodies specific for the test protein. The amount of antibody bound to the carrier is then evaluated. As a rule, primary or secondary antibodies labeled with a chemically bound enzyme are used for this [Voller, A., The enzyme-linked immunosorbent assay (ELISA) (theory, technique and applications). Ric Clin Lab, 1978. 8 (4): p. 289-98, de Savigny, D. and A. Voller, The communication of ELISA data from laboratory to clinician. J Immunoassay, 1980. 1 (1): p.105-28]. The enzyme associated with the antibody reacts with the appropriate substrate, changing its spectral characteristics as a result of the reaction, with the formation of a chemical moiety or compound that can be detected using spectrometric or fluoremetric methods, or visually. In addition, antibodies are also labeled with radioisotopes, metal nanoparticles and other substances. This principle is based on commercially available methods for the diagnosis of RMP, such as NMP-22 (detection in the urine of a nuclear protein released during apoptosis). This diagnostic test system can serve as a prototype of the present invention [Landman J, Chang Y, Kavaler E, Droller MJ, Liu BC., Sensitivity and specificity of NMP-22, telomerase, and BTA in the detection of human bladder cancer. Urology. 1998 Sep; 52 (3): 398-402]. The following criteria are used as diagnostic features in it: 1) a statistically significant increase in the level of marker content in the RMP sample relative to the upper limit of the norm; 2) pronounced dynamics of a decrease in marker levels with successful treatment of RMP by at least 50%. This system can also be used to detect relapses and predict the future course of the disease. At the same time, this and other accessible diagnostic systems for RMP, based on the detection of protein markers in physiological fluids, have insufficient sensitivity (less than 25%) for effective use in clinical practice.

The invention solves the problem of increasing the sensitivity of RMP markers and allows the diagnosis of RMP with a sensitivity of 93%.

The problem is solved by a method for the diagnosis of bladder cancer:

real-time PCR, including biomaterial production and RNA isolation, cDNA synthesis on the RNA matrix, normalization of the TRIB1 cDNA concentration by the control gene, quantitative PCR amplification of the TRIB1 gene fragment and diagnosis by determining the amount of the amplified TRIB1 DNA fragment for the biomaterial sample and a kit for the diagnosis of bladder cancer by real-time PCR, including primers with the sequence SEQ ID NO: 1 and 2 with a molar ratio of 1: 1;

In the present invention, products of the TRIB1 gene, which have high specificity of expression specifically in RMP cells, but not in normal bladder tissue, are used as a marker of RMP. Prior to the present invention, no published data on increased TRIB1 gene expression in cancer compared with normal bladder cells is available. In the present invention, as an indicator of the presence of cancerous and / or precancerous changes for RMP, a change in the RNA content of the TRIB1 gene in patients with RMP is provided.

During the search for differential transcripts by the authors using microarray analysis and identifying differentially expressed genes in normal and tumor samples of bladder tissue at different stages of malignant transformation, a significant increase in the mRNA content of the TRIB1 gene was detected already in the early stages of malignant transformation of bladder cells.

The present invention relates to a new marker for the diagnosis of bladder cancer (RMP), which is a mRNA gene TRIB1 and / or protein TRIB1. An increase in the gene mRNA content in presumably cancerous tissues of the human bladder compared with its content in normal / healthy tissues, as well as an increase in the content of TRIB1 protein (TRIB1 gene product) in the urine of a presumably sick human RMP compared to the protein content in the urine of a healthy person diagnostic marker RMP.

The present invention relates to a method for diagnosing bladder cancer. This method includes the following stages: obtaining initial samples of biomaterial, for example, tissues from a patient; Isolation and purification of RNA preparations from tissue samples; synthesis of cDNA on the RNA matrix, determination of the mRNA concentration of the TRIB1 gene using quantitative PCR amplification using a cDNA matrix; normalization of the mRNA concentration of the TRIB1 gene according to the control gene, the mRNA of which is relatively constant in healthy and cancerous human tissues, including the bladder; diagnostics of RMP. For example, an indication for the detection of RMP is the level of mRNA of the TRIB1 gene in the tissues of the bladder in excess of 4% of the level of mRNA in these tissues of the human beta-actin gene (ASTV).

Oligonucleotide primers used for the synthesis of single-stranded or double-stranded cDNA are selected from oligo (dT) -containing primers, random hexamers, or a combination thereof, as well as gene-specific primers.

For amplification of cDNA, oligonucleotide primers and a probe are used that are selected in such a way that they specifically hybridize with cDNA even in the presence of an impurity of genomic DNA. A possible sequence of primers is presented by SEQ ID NO 1-2.

The quantitative amplification reaction of the TRIB1 gene fragment is real-time PCR or RT-PCR.

The ASTV gene encoding actin beta is used as a control gene. A possible sequence of primers for determining the concentration of the control gene is presented by SEQ ID NO 3-4. The sequence of the protein-coding DNA of the TRIB1 gene is shown in SEQ ID NO 5. The sequence of the TRIB1 protein is shown in SEQ ID NO 6.

The set of primers for the implementation of the polymerase chain reaction for determining the content of mRNA of the TRIB1 gene have the sequence SEQ ID NO 1-2.

The present invention provides a variant of a method for the diagnosis of bladder cancer by real-time PCR based on measuring the mRNA content of the TRIB1 gene, as well as a kit for implementing this method. A reliably detectable difference in the mRNA content of the TRIB1 gene in normal and tumor tissues and / or blood of patients with RMP and healthy people can be used to detect RMP in the studied samples.

Biopsy samples, punctate, including material obtained by cystoscopy with direct biopsy, urine and peripheral blood are taken as samples for analysis.

Various methods can be used to isolate RNA. In classical RNA isolation methods, strong chaotropic agents are used, such as guanidinochloride and guanidinisothiocyanate, dissolving proteins, and sequential extraction with phenol and chloroform to denature and remove proteins [Sambrook J., Fritsch EF, Maniatis T., Molecular Cloning. A laboratory manual. 2 ^nd Edition ed. 1989, Cold Spring Harbor: CSHL Press]. The Trizol reagent method [GIBCO / Life Technologies] is also widely used.

In order to prevent RNA degradation by RNase enzymes, inhibitors are used, such as an RNAsin inhibitor of placental or recombinant origin or, for example, a vanadyl riboside complex, a non-specific broad-spectrum RNAse inhibitor.

Rapid and high-quality RNA isolation is also carried out using a number of commercially available kits (Clonogen, St. Petersburg; RNeasy kits (Qiagen); SV Total RNA Isolation System, Promega (USA), etc.). The use of various devices, for example, QuickGene-810 (Life Science, Japan), allows one to minimize work with aggressive agents and media, accelerate and simplify RNA isolation.

The reverse transcription reaction, as a result of which a single-stranded cDNA chain is synthesized on an RNA matrix, if necessary, with the completion of the second chain, allows us to switch from unstable RNA molecules to more stable DNA molecules. PCR amplification allows the use of small amounts of the original RNA (at the level of several nanograms), which corresponds to the minimum amounts of tissue under study. The reverse transcription reaction is carried out using commercially available reverse transcriptase preparations, such as Moloney mouse leukemia virus (M-MLV), avian myeloblastosis virus (AMV) reverse transcriptase, PowerScript (M-MLV-RT point mutation), C. Therm Polymerase, MINT -polymerase and others. For reverse transcription using different primers, for example:

1) Oligo (dT) n-containing primers bind to the poly (A) tail at the 3'-end of the mRNA (the number n is usually 12-18, but can reach a larger value). These primers are used to obtain full-size cDNA.

2) Sets of random hexanucleotide primers (statistical primers) hybridize with RNA in numerous sites. When reverse transcription with these primers receive cDNA, shortened relative to the size of the original RNA. Random hexamers are used to overcome the difficulties associated with reverse transcriptase passage of the secondary RNA structure; they are more effective in reverse transcription of 5'-regions of mRNA.

3) Hexamers or other short oligonucleotides of random composition (up to 12 nucleotides) can be used in combination with oligot-containing primers;

4) Specific oligonucleotide primers are used for reverse transcription of the mRNA region of interest for the study.

Gene transcription analysis is performed using cDNA as a template for quantitative PCR.

For the selection of specific primers and probes, special programs are used, many of which are freely available on the Internet. Among these programs are Oligo (version 6.42), PrimerSelect from the Lasergene package (www.dnastar.com). Primer Express (Applied Biosystems, USA), Primer Designer (IMB), FastPCR (http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm), PrimerQuest (http://scitools.idtdna.com/ Primerquest /), etc., as well as programs such as Vector NTI (Informax), Gene Runner, etc. In addition, it is possible to select primers without using special software.

In a preferred embodiment, primers with the sequence SEQ ID NO 1-2 are used.

All variants of detection of PCR products can be divided into specific and non-specific to a specific DNA sequence. Non-specific systems can be divided into systems using intercalating dyes and systems with primer labeling with fluorescent dyes.

The most inexpensive, but at the same time highly sensitive system is PCR in the presence of intercalating dyes, for example ethidium bromide, YOYO [Srinivasan, K., S.C. Morris, J.E. Girard, et al., Enhanced detection of PCR products through use of TOTO and YOYO intercalating dyes with laser induced fluorescence-capillary electrophoresis. Appl Theor Electrophor, 1993, 3 (5): p.235-9], YO-PRO-1 [Ishiguro, T., J. Saitoh, H. Yawata, et al., Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent intercalater. Anal Biochem, 1995, 229 (2): p.207-13], SYBR Green I [Morrison, T.B., J.J. Weis and C.T. Wittwer, Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques, 1998, 24 (6): p. 954-8, 960, 962], SYBR Gold, Eva Green and others. These dyes integrate into a double-stranded DNA molecule, change the spectral characteristics of the dyes, which is reflected in a change in the fluorescent signal, amplified by as PCR product accumulates.

High specificity of PCR-RV can be achieved by using a probe (TaqMan system) containing a fluorophore or fluorescent dye at the 5'-end (for example, FAM - 6-carboxy-fluoroscein), and at the 3'-end - the so-called quencher (e.g. DABCYL - 4- (dimethylammoazo) benzene-4-carboxylic acid). During PCR, the interaction of FAM and DABCYL is disrupted due to the cleavage of the Taq probe with DNA polymerase due to its 5'-exonuclease activity, and fluorescence emission recorded by the device occurs [Holland, PM, RDAbramson, R. Watson, et al., Detection of specific polymerase chain reaction product by utilizing the 5 '---- 3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA, 1991, 88 (16): p. 7276-80]. The selection of probes for PCR-RV is carried out in accordance with standard recommendations and the requirements of the method (protocols of Applied Biosystems, http://docs.appliedbiosystems.com). In addition, other modifications of this method can be used, such as displacing probes, molecular beacons, adjoining samples [Solinas, A., LJBrown, C. McKeen, et al., Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res, 2001.29 (20): p.E96, Li, Q., G. Luan, Q. Guo, et al., A new class of homogeneous nucleic acid probes based on specific displacement hybridization. Nucleic Acids Res, 2002, 30 (2): p.E5, Tyagi, S. and F.R. Kramer, Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol, 1996, 14 (3): p.303-8, Didenko, V.V., DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. Biotechniques, 2001, 31 (5): p. 1106-16, 1118, 1120-1].

A quantitative assessment of the mRNA content is achieved using parallel PCR-RV with the test and control samples (Figure 2). As an internal control, relative to which the amplification products of the studied TRIB1 gene are normalized, the “housekeeping gene”, ASTV, encoding the beta-actin basic protein of the cytoskeleton, is selected. According to the preferred form of quantifying the mRNA content, it is necessary to select a control gene having a low variability of the mRNA content in tumor and normal tissues of the bladder compared to the variability of the mRNA content of the studied genes [Radonic A., Thuike S., Mackay IM, Landt O., Siegert W., Nitsche A. 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313, 856-862]. As a rule, “housekeeping” genes, expressed at a high level in each cell of the body, are chosen as control genes, although any gene with a relatively constant mRNA content in the studied samples can be used for this purpose. The decision to select a gene as a control depends on the degree of accuracy selected / required. In a preferred embodiment of the present invention, a traditional and frequently used control gene for ASTV is selected.

Estimation of the mRNA content of genes can be based on the absolute and relative measurement of the number of copies of the studied transcripts - the absolute method (standard curve method) and the relative measurement method (ΔΔСt - method, http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf). According to a preferred form of quantifying the mRNA content of genes, the second of them is selected as the main measurement method. This method allows a double comparison of the data for the studied and control genes in the tumor and normal and does not require alignment of the concentrations of tumor and normal RNA / cDNA samples, which is necessary when using other methods, for example, RT-PCR.

According to a preferred form of quantifying the mRNA content of the genes, it is important to verify the suitability of the reference samples, in this case conditional norms. A “conventional norm” is considered to be a sample of bladder tissue with missing macro- and microscopic signs of tumor growth or a sample of venous blood of a patient. In addition, additional comparison samples obtained from conditionally healthy donors after death (donors who did not suffer bladder cancer during their lifetime) were used.

Since not all tumor samples had suitable paired conditional norms, the calculations of the relative mRNA content of the TRIB1 gene were carried out using different comparison samples — paired conditional norms, if any, and norms from conditionally healthy donors, if there were no paired norms.

The invention is illustrated by graphic materials.

Figure 1. Schematic representation of the structure of the gene TRIB1, cDNA TRIB1 and the resulting amplification product using PCR and / or PCR-RV.

Figure 2. Electrophoretic separation in a 1.5% agarose gel of the PCR-PB product (35 PCR cycles) - TRIB1 gene transcript (size 158 bp), 1st and 2nd tracks - TRIB1 gene transcript of the expected size in tumor tissues, the 3rd and 4th paths - in normal tissues of the bladder, the 5th path - a marker of molecular masses of DNA from 100 to 1500 bp

Figure 3. Relative level of TRIB1 gene expression in tumor and normal bladder tissues, measured by PCR-RV after normalizing its expression relative to ASTV gene expression in the same tissue samples. Samples No. 1-9 - tumor tissue of the bladder, 10-12 - normal (without malignant transformation) tissue of the bladder.

The invention is illustrated by examples.

Example 1. Determination of the mRNA level of the TRIB1 gene in normal and cancerous tissues of the bladder.

1) Tissue samples

Tissue samples of different histological types of PRMP (T), morphologically normal tissues adjacent to tumors (the so-called conditional norms (N), as well as normal tissues of the bladder (NB) from suddenly died donors were collected and characterized independently by two different groups of FSI employees MNIII named after P.A. Herzen of Rosmedtechnology and the faculty of fundamental medicine of Moscow State University named after M.V. Lomonosov). The clinical diagnosis was established taking into account data from a retrospective analysis of the primary medical documentation of patients, cytological and histological studies of biopsy specimens obtained by cystoscopy, as well as samples of bladder tissue obtained after transurethral resection and surgical material. Bladder tissue samples (tumor, conditionally normal tissue) were obtained immediately after removal of the tumor. Each sample was stored in an RNALater Reagent (QIAGEN) solution stabilizing RNA molecules at a temperature of -70 ° C. All tumor samples were characterized according to the international system of clinical and morphological classification of TNM tumors, where T (tumor) - T0-T4 - categories reflecting an increase in the size and / or local distribution of the primary tumor, N (nodules) - N0-N3 - categories reflecting different the degree of defeat by metastases of the regional lymph nodes; M (metastasis) - M0-M1 - characterizes distant metastases. All possible combinations of TNM are combined into larger groups - stages that reflect the course of the tumor process.

2) Isolation of RNA preparations from tissues

Total RNA was isolated from frozen, stored in RNALater Reagent (QIAGEN) solution samples of tumor and normal tissue using TRIZOL Reagent (Invitrogen). The main steps include:

a) the destruction of tissue frozen in liquid nitrogen, using a microdesembler ("Sartorius", Germany) and the homogenization of the destroyed sample in a lysis solution containing guanidine isothiocyanate and phenol;

b) the addition of chloroform and centrifugation for 10 minutes at ≤12000 × g at a temperature of 4 ° C;

c) precipitation of RNA from the aqueous phase formed after centrifugation by adding isopropyl alcohol and centrifuging again for 10 minutes at ≤12000 × g at 4 ° C.

Next, the RNA pellet after repeated centrifugation is washed with aqueous 75% ethanol, dried and dissolved in water purified from nucleases.

The concentration of RNA is determined spectrophotometrically. The quality of the isolated RNA was checked by electrophoresis in 1% agarose in the presence of ethidium bromide and spectrophotometrically (Nanodrop, USA).

3) reverse transcription reaction

Single-stranded cDNA is synthesized on an RNA template isolated as described in paragraph 2). For the reverse transcription reaction, 1 μg of RNA obtained by one of the two methods described above using Trizol Reagent reagent kits (Invitrogen) or RNeasy Mini Kit reagents (Qiagen) pretreated with RNase-free DNase I (Sigma-Aldrich) and incubated 5 is taken. min at 70 ° C, then placed in ice.

For each sample, 20 μl of a mixture containing:

5 mM MgCl ₂ ;

1 × AMV reverse transcriptase buffer containing 250 mM Tris-HCl (pH 8.3, 25 ° C), 250 mM KCl, 50 mM MgCl ₂ , 2.5 mM spermidine and 50 mM DTT (Promega);

100 ng hexanucleotide primers;

1 mM dNTP (Evrogen);

200 units of AMV reverse transcriptase (Promega).

RNA and water are added to the mixture to a final volume of 20 μl and the reaction is carried out at the following temperature conditions: 25 ° C for 10 minutes, 42 ° C for 60 minutes, 70 ° C for 10 minutes.

4) Selection of conditions for determining the mRNA content of the TRIB1 gene in tissue samples of the bladder

For RT-PCR and PCR-PB, the same primers TRIB1_F (SEQ ID NO: 1) and TRIB1_R (SEQ ID NO: 2) are used with their molar ratio 1: 1, specific for different exons of the TRIB1 gene, one of the primers overlapping the boundary between exons. Amplicon size - 158 bp

The sequence of the selected primers for the control gene: SEQ ID NO 3-4, with their molar ratio of 1: 1, the size of the amplicon is 145 bp

For PCR-PB select the optimal concentration of primers of the studied and control genes (Figure 3).

5) Quantification of the mRNA content of the TRIB1 gene

For quantitative measurements using the device Stratagene MX3000P (USA).

Protocol for determining the mRNA content by PCR-RV using the kit “Set of reagents for PCR-RV in the presence of SYBR Green I” or “Set of reagents for PCR-RV in the presence of EVA Green” (Syntol, Russia):

1. Prepare the reaction mixtures for the TRIB1 and ASTV genes, carefully mixing all the components of the reaction, except the template (cDNA), at the rate of 1 reaction with a volume of 25 μl for each sample +1 additional reaction of 25 μl.

Table 1 The composition of the reaction mixture for the ASTV gene: Reagent The concentration of stock solutions Volume per reaction (μl) The final concentration in the reaction PCR Buffer B 10X 2.5 1X dNTPs 2.5 mm 2.5 250 μM MgCl ₂ 25 mm 2.5 2.5 mm Primer ACTB_F 10 μM one 0.4 μM Primer ACTB_R 10 μM one 0.4 μM Taq DNA polymerase 5 units / μl 0.5 0.1 units / μl H ₂ O 13,2 cDNA 2

table 2 The composition of the reaction mixture for the TRIB1 gene: Reagent The concentration of stock solutions Volume per reaction (μl) The final concentration in the reaction PCR Buffer B 10X 2.5 1X dNTPs 2.5 mm 2.5 250 μM MgCl ₂ 25 mm 2.5 2.5 mm Primer TRIB1_F 10 μM one 0.4 μM Primer TRIB1_R 10 μM one 0.4 μM Taq DNA polymerase 5 units / μl 0.5 0.1 units / μl H ₂ O 13,2 cDNA 2

2. In 0.2 ml tubes designed for PCR-PB, add 23 μl of the prepared reaction mixtures without a matrix.

3. Pipette 2 μl of the matrix, tightly close the tubes with caps.

4. Place the tubes in the instrument compartment, set the cell names, temperature conditions for 35 cycles: 95 ° С - 5 min - denaturation and enzyme activation, 95 ° С - 20 sec - denaturation, 60 ° С - 20 sec - probe annealing and polymerization , 72 ° С - 30 sec - elongation of chains.

5. The reactions are carried out in the mode of relative quantitative measurements (software Stratagene MxPro, USA).

6. After completion of the amplification reaction, data is saved.

7. The reaction products are analyzed by gel electrophoresis in TBE buffer containing 10 mg / l ethidium bromide. For this, 5 μl of amplification products (40 cycles) are taken, 1.8% agarose gel containing 0.5 mg / L ethidium bromide samples and a molecular weight marker for DNA are used to evaluate the size of the amplification products (SibEnzyme NPO).

6) Mathematical processing of PCR-RV data

PCR-RV data is transferred as a text file to Microsoft Excel and mathematical processing of the data is carried out. The relative content of mRNA of the TRIB1 - R _{cDNA gene} (hereinafter R) in tumor and normal tissues of the bladder is normalized relative to the content of mRNA of the ASTV gene according to the formula:

R = 2 ^ (Ct (ACTB) -Ct (TRIB1), where Ct is the threshold cycle.

The range of extreme values of R is calculated taking into account the calculated deviations E for the Ct values:

E = ((Σ (x-x _avg ) ² ) / (n-1)) ^1/2 ,

where x - values obtained as a result of measurement (number 1, number 2 ...),

x _cp = Σx / n,

n is the sample size (the number of repetitions of the reaction).

The reliability of the observed changes is estimated based on the normal distribution of data. Data is considered reliable at P <0.05, where P is an indicator of the statistical significance of the data (Figure 4).

7) Results

As a result, for all the studied samples of RMP (n = 26), a 7-83-fold excess of the mRNA level of the TRIB1 gene was revealed relative to normal samples of bladder tissue (n = 47)

Example 2. Determination of the concentration of mRNA of the TRIB1 gene in the urine and blood of patients with RMP and healthy people.

1) Urine and blood samples

Blood and urine samples from patients diagnosed with RMP, as well as from healthy donors, were collected and characterized by a group of employees of FGU MNIII im. P.A. Herzen of the Russian Medical Technologies. The clinical diagnosis was established taking into account data from a retrospective analysis of the primary medical documentation of patients, cytological and histological studies of biopsy specimens obtained by cystoscopy, as well as samples of bladder tissue obtained after transurethral resection and surgical material. Blood is mixed with a solution of an anticoagulant (for example, sodium citrate, EDTA, etc.) in a ratio of 9: 1 (9 parts of blood and 1 part of an anticoagulant), the plasma is separated from blood cells by centrifugation for 15 minutes at 1500 × g and immediately mixed with a solution that stabilizes RNA molecules (RNAlater Reagent, QIAGEN). The resulting solution is stored at a temperature of -20 ° C. Urine samples are mixed with a solution of RNAlater Reagent, QIAGEN, frozen and stored at a temperature of -20 ° C.

2) Isolation of RNA preparations from tissues

Total RNA was isolated from frozen, stored in a solution of RNALater Reagent (QIAGEN) urine and blood samples obtained from patients and healthy donors using TRIZOL Reagent (Invitrogen). The main steps include:

a) Treatment of blood and urine with a lysing solution containing guanidine isothiocyanate and phenol;

b) the addition of chloroform and centrifugation for 10 minutes at 12000 × g at a temperature of 4 ° C;

c) precipitation of RNA from the aqueous phase formed after centrifugation by adding isopropyl alcohol and centrifuging again for 10 minutes at 12000 × g at 4 ° C.

Next, the RNA pellet after repeated centrifugation is washed with aqueous 75% ethanol, dried and dissolved in water purified from nucleases.

The concentration of RNA is determined spectrophotometrically. The quality of the isolated RNA was checked by electrophoresis in 1% agarose in the presence of ethidium bromide and spectrophotometrically (Nanodrop, USA).

3) reverse transcription reaction

Single-stranded cDNA is synthesized on an RNA template isolated as described in paragraph 2). For the reverse transcription reaction, 1 μg of RNA obtained by one of the two methods described above using Trizol Reagent reagent kits (Invitrogen) or RNeasy Mini Kit reagents (Qiagen) pretreated with RNase-free DNase I (Sigma-Aldrich) and incubated 5 is taken. min at 70 ° C, then placed in ice.

For each sample, 20 μl of a mixture containing:

5 mM MgCl ₂ ;

1 × AMV reverse transcriptase buffer containing 250 mM Tris-HCl (pH 8.3, 25 ° C), 250 mM KCl, 50 mM MgCl ₂ 2.5 mM spermidine and 50 mM DTT (Promega);

100 ng hexanucleotide primers;

1 mM dNTP (Evrogen);

200 units of AMV reverse transcriptase (Promega).

RNA and water are added to the mixture to a final volume of 20 μl and the reaction is carried out at the following temperature conditions: 25 ° C for 10 minutes, 42 ° C for 60 minutes, 70 ° C for 10 minutes.

4) Selection of conditions for determining the content of mRNA of the TRIB1 gene

For RT-PCR and PCR-PB, the same primers TRIB1_F (SEQ ID NO: 1) and TRIB1_R (SEQ ID NO: 2) are used, specific for different exons of the TRIB1 gene, with their molar ratio 1: 1, one of the primers overlapping the border between exons. Amplicon size - 158 bp

The sequence of the selected primers for the control gene: SEQ ID NO 3-4, the size of the amplicon - 145 BP

For PCR-RV, optimal primer concentrations of the studied and control genes are selected.

5) Quantification of the mRNA content of the TRIB1 gene

For quantitative measurements using the device Stratagene MX3000P (USA).

Protocol for determining the mRNA content by PCR-RV using the kit “Set of reagents for PCR-RV in the presence of SYBR Green I” or “Set of reagents for PCR-RV in the presence of EVA Green” (Syntol, Russia):

1. Prepare the reaction mixtures for the TRIB1 and ASTV genes, carefully mixing all the components of the reaction, except the template (cDNA), at the rate of 1 reaction with a volume of 25 μl for each sample +1 additional reaction of 25 μl.

Table 3 The composition of the reaction mixture for tesh ASTV: Reagent The concentration of stock solutions Volume per reaction (μl) The final concentration in the reaction PCR Buffer B 10X 2.5 1X dNTPs 2.5 mm 2.5 250 μM MgCl ₂ 25 mm 2.5 2.5 mm Primer ACTB_F 10 μM one 0.4 μM Primer ACTB_R 10 μM one 0.4 μM Taq DNA polymerase 5 units / μl 0.5 0.1 units / μl H ₂ O 13,2 cDNA 2

Table 4 The composition of the reaction mixture for the TRIB1 gene: Reagent The concentration of stock solutions Volume per reaction (μl) The final concentration in the reaction PCR Buffer B 10X 2.5 IX dNTPs 2.5 mm 2.5 250 μM MgCl ₂ 25 mm 2.5 2.5 mm Primer TRIB1_F 10 μM one 0.4 μM Primer TRIB1_R 10 μM one 0.4 μM Taq DNA polymerase 5 units / μl 0.5 0.1 units / μl H ₂ O 13,2 cDNA 2

2. In 0.2 ml tubes designed for PCR-PB, add 23 μl of the prepared reaction mixtures without a matrix.

3. Pipette 2 μl of the matrix, tightly close the tubes with caps.

4. Place the tubes in the instrument compartment, set the cell names, temperature conditions for 35 cycles: 95 ° С - 5 min - denaturation and enzyme activation, 95 ° С - 20 sec - denaturation, 60 ° С - 20 sec - probe annealing and polymerization , 72 ° С - 30 sec - elongation of chains.

5. The reactions are carried out in the mode of relative quantitative measurements (software Stratagene MxPro, USA).

6. After completion of the amplification reaction, data is saved.

7. The reaction products are analyzed by gel electrophoresis in TBE buffer containing 10 mg / l ethidium bromide. For this, 5 μl of amplification products (40 cycles) are taken, 1.8% agarose gel containing 0.5 mg / L ethidium bromide samples and a molecular weight marker for DNA are used to evaluate the size of the amplification products (SibEnzyme NPO).

6) Mathematical processing of PCR-RV data

PCR-RV data is transferred as a text file to Microsoft Excel and mathematical processing of the data is carried out. The relative content of mRNA of the TRIB1 - R _{cDNA gene} (hereinafter R) in tumor and normal tissues of the bladder is normalized relative to the content of mRNA of the ASTV gene according to the formula:

R = 2 ^ (Ct (ACTB) -Ct (TRIB1), where Ct is the threshold cycle.

The range of extreme values of R is calculated taking into account the calculated deviations E for the Ct values:

E = ((Σ (x-x _avg ) ² ) / (n-1)) ^1/2 ,

where x - values obtained as a result of measurement (number 1, number 2 ...),

x _cf = Σx / n,

n is the sample size (the number of repetitions of the reaction).

The reliability of the observed changes is estimated based on the normal distribution of data. Data is considered reliable at P <0.05, where P is an indicator of the statistical significance of the data.

7) Results

As a result, for all investigated urine and blood samples obtained from patients with RMP (n = 26), an 11-75-fold excess of the mRNA concentration of the TRIB1 gene was revealed relative to urine and blood samples obtained from healthy donors (n = 62).

Example 3

Evaluation of the effectiveness of the treatment of RMP by changing the level of TRIB1 gene expression in the urine and / or blood of the examined patients.

Evaluation of the effectiveness of the treatment of RMP is carried out by analyzing the level of TRIB1 gene expression in the urine and / or blood of patients with RMP before treatment and after the treatment by the method specified in example 2. As a result, for all examined patients (n = 12) with a diagnosis of RMP revealed a decrease in the level of TRIB1 gene expression in urine and blood by 2-8 times after the treatment compared with the initial level of expression of this gene in urine and blood detected before treatment. This indicates the possibility of using the present invention in monitoring the effectiveness of anti-cancer therapy, and analysis by the methods of the present invention should be carried out before and after the course of treatment, and also, if necessary, during the course of treatment. A decrease in the mRNA content of the TRIB1 gene during or at the end of the course of treatment may indicate positive changes in treatment. A further increase in the mRNA content of the TRIB1 gene indicates a low treatment efficiency.

Claims (2)

1. A method for the diagnosis of bladder cancer by real-time PCR, including biomaterial production and RNA isolation, cDNA synthesis on an RNA matrix, normalization of the TRIB1 cDNA concentration by the control gene, quantitative PCR amplification of the TRIB1 gene fragment and diagnosis by determining the concentration of the amplified fragment TRIB1 DNA in the test sample and its comparison with the concentration of the amplified TRIB1 DNA fragment from a healthy donor, and when the concentration of the amplified DNA fragment is exceeded Bladder cancer is diagnosed with TRIB1 in the test sample compared to that obtained from a healthy donor 11-75 times. 2. The kit for the diagnosis of bladder cancer according to claim 1 by real-time PCR, including primers with the sequence of SEQ ID NO: 1 and 2 with a molar ratio of 1: 1.

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