RU2798295C1 - Set of housekeeping genes for analysis of gene expression in parkinson's disease in various brain and peripheral blood tissues of mice and its application - Google Patents

Abstract

FIELD: genetics.

SUBSTANCE: method for analyzing gene expression in Parkinson's disease is described. It involves the isolation of RNA from samples of brain tissue and peripheral blood and the use of a set of primers and probes to determine the stability of the expression of a set of housekeeping genes. The set consists of at least 3 housekeeping genes selected from the group consisting of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, in brain tissue and peripheral blood of mice.

EFFECT: determination of housekeeping genes for the analysis of gene expression in Parkinson's disease, showing the validity and stability of expression in various tissues of the brain and peripheral blood of mice.

11 cl, 1 dwg, 5 tbl, 1 ex

Description

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of genetics. In particular, the present invention relates to the use of a housekeeping set consisting of at least 3 housekeeping selected from the group consisting of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, for the analysis of gene expression in Parkinson's disease, where the specified set housekeeping is stably expressed in various tissues of the brain and peripheral blood of mice, to a set of primers and probes for determining the stable expression of the specified set of housekeeping, as well as to a method for analyzing gene expression in Parkinson's disease using the specified set of housekeeping.

Sequence listing

This application contains a sequence listing that has been filed on an electronic medium in a machine-readable format and is hereby incorporated herein by reference in its entirety.

Inclusion by reference

The background to the creation of the present invention was disclosed by the authors of the present invention earlier in the publication of Alieva, Anelya Kh, et al. "Housekeeping Genes for Parkinson's Disease in Humans and Mice" Cells, 2021. 10(9): 2252 published August 30, 2021 , since this application for a patent for an invention was filed with the federal executive authority for intellectual property before the expiration of six months from the date of disclosure of information. All other publications, patents, and patent applications referred to in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and separately identified as being incorporated by reference.

Background of the Invention

Parkinson's disease (PD) is an age-dependent neurodegenerative disease characterized by selective death of dopaminergic neurons in the compact substantia nigra with the accumulation of an abnormal form of the synaptic protein α-synuclein in them, as well as pathology of the nigrostriatal dopaminergic pathway. Decreased striatal dopamine levels in PD result in a number of movement disorders such as bradykinesia. muscle rigidity and others. The prevalence of PD, disability of patients, high financial costs for treatment and rehabilitation make this disease socially significant.

An important feature of this disease is a long latent period, and therefore it is necessary to search for prognostic biomarkers. One of the methods for detecting biomarkers of PD is the study of changes in gene expression in the peripheral blood and tissues of patients at an early stage of the disease and not undergoing treatment.

To date, a number of genes have been identified, the change in the expression of which makes it possible to detect and/or predict PD.

It is known that in the onset of PD, the process of damage to dopaminergic structures proceeds for a long time without obvious clinical manifestations, which is probably due to the inclusion of various compensatory mechanisms. Symptoms of the disease occur only at a fairly "advanced" stage of neuropathology, when more than 50% of dopaminergic neurons in the substantia nigra are involved in degeneration, and the level of dopamine in the striatum decreases by more than 70%. Obviously, this imposes serious restrictions on the ability to provide effective care to patients. That is why the efforts of researchers are currently focused on the development of preclinical diagnostics and preventive treatment of PD.

For example, international patent publication WO 2005/067391 A2 discloses molecular markers for detecting, predicting and monitoring PD, where said molecular markers represent one or more genes with an altered expression pattern.

Active study of gene expression continues at the present time. The simplest and most widely used method for analyzing gene expression at the mRNA level is a semi-quantitative approach based on the reverse transcription reaction (RT) and quantitative real-time PCR (qPCR). This method is considered the gold standard for studying changes in gene expression at the mRNA level in biomedical research. A critical aspect of this method, as with other semi-quantitative methods, is the availability of reference genes as an internal control to normalize expression data for a candidate gene. Based on this, it can be concluded that the exact determination of changes in the expression of candidate genes depends on the correct choice of internal reference genes. Such genes are called housekeeping.

A number of documents are known from the prior art that disclose the use of housekeeping genes to normalize expression data for a candidate gene, the change in expression of which is associated with the development of PD.

In Morató, Xavier, et al. "Ecto-GPR37: a potential biomarker for Parkinson's disease." Translational neurodegeneration, 2021, 10(1): 1-14, describes a technique for real-time PCR (RT-qPCR) in the analysis of gene expression in PD in the brain tissues and cerebrospinal fluid of mice and humans.

A similar real-time PCR (RT-qPCR) technique for analyzing gene expression in PD is described in Garcia Esparcia, Paula, "Identification of a risk transcriptome and proteome in Parkinson's disease, Dementia with Lewy bodies and rapidly progressive Dementia with Lewy bodies", Doctoral Thesis in Biomedicine specialty Neuroscience School of Medicine, Barcelona, September 2016, which also used a set of Housekeeping Gep to normalize expression data for the candidate gene.

US 8257929 B2 discloses studies of changes in gene expression associated with PD by examining multi-regional gene expression in the brain without pathology and the brain with PD. This document also defines changes in gene expression associated with PD. by studying gene expression in normal blood and blood of patients with PD using a set of housekeeping genes to normalize expression data for a candidate gene.

US 2015/086481 A1 discloses an analysis of GUCY2C gene expression in the brain in PD. To do this, real-time PCR (RT-qPCR) was performed to enrich the corresponding RNA of each line, also using housekeeping genes to normalize data transcripts.

In Renton, Alan Edward Mayo, "Gene expression and genetic analyzes in Parkinson's disease with and without dementia". Thesis for the degree of Doctor of Philosophy to University College London Department of Molecular Neuroscience Institute of Neurology Institute of Human Genetics and Health University College London, 2010, disclosed a technique for analyzing target gene mRNA expression in several brain regions, also using housekeeping genes for normalization data on the expression of candidate genes.

It is important to note that in all the cited sources, genes already known for use as housekeeping genes were used as housekeeping genes without conducting studies to determine the stability of the expression of the selected housekeeping genes. However, the sources cited confirm the necessary and important importance of housekeeping genes for accurately determining changes in the expression of candidate genes associated with PD.

Another important step in this area is the development of models of preclinical and early clinical stages of PD in animals, which will not only provide new fundamental knowledge about the pathogenesis of the disease, but will also contribute to the search for new therapeutic strategies.

To study the experimental aspects of PD, well-established toxic models on laboratory animals, mainly on rodents, are used. One of the most commonly used is the model in which BP is induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). This toxin is administered systemically, easily penetrates the blood-brain barrier, which leads to symmetrical damage to dopaminergic structures and quite accurately reflects the nature of pathological changes in the central nervous system that are observed in patients with PD.

At the same time, it is obvious that, in addition to the development of models of preclinical and early clinical stages of PD in animals, the identification of housekeeping genes with stable expression for these models is of great importance.

However, it is known that the expression of the most common housekeeping can depend on external conditions and change in pathologies. Significant disadvantages of using such housekeeping in research are the lack of their validation in specific experimental conditions, as well as the blind transfer of housekeeping. selected for one organism to another, without concomitant careful checking. This, in turn, can affect the results obtained and/or lead to false results in assessing changes in the expression of candidate genes.

In this regard, there remains a need in the art to determine housekeeping genes that show the validity and stability of expression in mice with the pathology under study.

Brief summary of the present invention

Based on the shortcomings of the prior art, the task of the present invention, consisting in the detection of housekeeping genes showing the validity and stability of expression in the studied objects with the studied pathology, was solved by the authors of the present invention by detecting a specific set of housekeeping for analysis of the expression of genes in Parkinson's disease, stably expressed in various tissues of the brain and peripheral blood of mice.

Thus, one object of the present invention is the use of a set of housekeeping, consisting of at least 3 housekeeping selected from the group consisting of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, for the analysis of gene expression in Parkinson's disease, where the specified a set of housekeeping stably expressed in various tissues of the brain and peripheral blood of mice.

According to one embodiment of the present invention, the set of housekeeping consists of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars and is stably expressed in various tissues of the brain and peripheral blood of mice.

According to another embodiment of the present invention, the brain tissues are the brain as a whole.

According to another embodiment of the present invention, brain tissues are selected from the group consisting of cortex, cerebellum, striatum and substantia nigra.

According to another embodiment of the present invention, the set of housekeeping consists of housekeeping Aars, Psmd7 and Psmd6 and is stably expressed in the whole brain.

According to another embodiment of the present invention, the housekeeping set consists of Polr2f Aars and Bcat2 housekeeping and is stably expressed in the cortex.

According to another embodiment of the present invention, the housekeeping set consists of Psmd6, Aars and Polr2f housekeeping and is stably expressed in the cerebellum.

According to another embodiment of the present invention, the housekeeping set consists of Psmd7, Aars and Sars housekeeping and is stably expressed in the striatum.

According to another embodiment of the present invention, the housekeeping set consists of Psmd7, Aars and Bcat2 housekeeping and is stably expressed in substantia nigra.

According to another embodiment of the present invention, the set of housekeeping consists of Polr2f, Hprt and Sars housekeeping and is stably expressed in peripheral blood.

Another object of the present invention is a set of primers and probes for determining the stable expression of a housekeeping set. consisting of at least 3 housekeeping selected from the group consisting of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, in various tissues of the brain and peripheral blood of mice, where the set of primers and probes is:

Another object of the present invention is a method for analyzing gene expression in Parkinson's disease. involving the use of a set of housekeeping. consisting of at least 3 housekeeping selected from the group consisting of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, in various tissues of the brain and peripheral blood of mice using the specified set of primers and probes.

According to one embodiment of the claimed method, the housekeeping set consists of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars and is stably expressed in various tissues of the brain and peripheral blood of mice.

According to another embodiment of the claimed method, the brain tissue is the brain as a whole.

According to another embodiment of the claimed method, brain tissues are selected from the group consisting of cortex, cerebellum, striatum and substantia nigra.

According to another embodiment of the claimed method, the set of housekeeping consists of housekeeping Aars, Psmd7 and Psmd6 and is stably expressed in the brain as a whole.

According to another embodiment of the claimed method, the set of housekeeping consists of housekeeping Polr2f, Aars and Bcat2 and is stably expressed in the cortex.

According to another embodiment of the claimed method, the housekeeping set consists of Psmd6, Aars and Polr2f housekeeping and is stably expressed in the cerebellum.

According to another embodiment of the claimed method, the housekeeping set consists of Psmd7, Aars and Sars housekeeping and is stably expressed in the striatum.

According to another embodiment of the claimed method, the set of housekeeping consists of housekeeping Psmd7, Aars and Bcat2 and is stably expressed in substantia nigra.

According to another embodiment of the claimed method, the set of housekeeping consists of Polr2f, Hprt and Sars housekeeping and is stably expressed in peripheral blood.

Brief description of the drawings

In FIG. 1 shows an example of data processing for analysis of relative mRNA levels. The RefFinder software calculates reference gene scores using the geNorm, Normfinder, BestKeeper algorithms and the ΔΔCt comparison method, as well as the Comprehensive Ranking value. Based on these scores, the program assigns each gene an appropriate score and calculates the geometric mean of their weights for the overall final ranking. The program selects the lowest score among the ranks assigned by each algorithm separately, and then assigns the final overall score. The closer the final score is to one, the more stable housekeeping is considered.

Detailed disclosure of the present invention

Definitions

In the context of the present invention, the term "housekinning" or "MKS" means an internal reference gene that acts as an internal control for the normalization of expression data for a candidate gene. As HKG, genes with stable expression in all biological samples are most often considered, and whose expression does not change under experimental conditions or under disease conditions. Housekeeping are genes that control basic metabolism and are responsible for cell survival, maintenance of cell homeostasis, and/or other fundamental processes in the cell.

In the context of the present invention, the term "candidate gene" means a gene. the change in the expression of which should be detected using normalization relative to the expression of the corresponding housekeeping.

In the context of the present invention, the term "primer" means an oligonucleotide capable of acting as an initiation point for the synthesis of a primer extension product, which is a complementary strand of a nucleic acid (of all types of DNA or RNA), under suitable amplification conditions (for example, buffer, salt, temperature and pH ) in the presence of nucleotides and an agent that polymerizes nucleic acids (for example, a DNA-dependent or RNA-dependent polymerase).

In the context of the present invention, the term "forward primer" means a primer that hybridizes (or anneals) to a target sequence (eg, template strand). The phrase "reverse primer" means a primer that hybridizes (or anneals) to a complementary strand of a target sequence. The forward primer hybridizes closer to the 5' end of the target sequence than the reverse primer.

In the context of the present invention, the term "probe" means an oligonucleotide capable of selectively hybridizing to at least a portion of a target sequence under appropriate amplification conditions (eg, an amplified portion of a target sequence).

In the context of the present invention, the term "set of primers and probes" means a combination comprising two or more primers. which together serve as a primer for amplifying the target sequence or target nucleic acid and at least one probe capable of detecting the target sequence or target nucleic acid. In general, the probe hybridizes to the amplification product (or amplicon) strand, resulting in an amplification product-probe hybrid that can be detected using standard methods known in the art.

In the context of the present invention, the terms "target sequence" or "target nucleic acid" are used interchangeably and mean the entity whose presence or absence is to be detected.

Disclosure of the present invention

Currently, there is an active study of gene expression. The simplest and most widely used method for analyzing gene expression at the mRNA level is a semi-quantitative approach based on the reverse transcription reaction (RT) and quantitative real-time PCR (qPCR). This method is considered the gold standard for studying changes in gene expression at the mRNA level in biomedical research. A critical aspect of this method, as with other semi-quantitative methods, is the availability of reference genes as an internal control to normalize expression data for a candidate gene. Based on this, it can be concluded that the exact determination of changes in the expression of candidate genes depends on the correct choice of internal reference genes. Such genes are called housekeeping.

However, the prior art does not disclose data from studies aimed at confirming the validity and stability of the expression of housekeeping for the analysis of gene expression in Parkinson's disease in mice, in particular in various tissues of the brain and peripheral blood of mice.

In order to solve the problem of the present invention, the authors of the present invention carried out a search, selection and study of the most stable housekeeping for the analysis of gene expression in Parkinson's disease in mice, in particular in various tissues of the brain and peripheral blood of mice. The studies carried out by the authors of the present invention concerned the determination of the stability of housekeeping in the main tissues of the mouse brain, as one of the main objects of modeling Parkinson's disease. In this case, a toxic model was chosen based on the administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). In particular, the authors of the present invention used for analysis a model of early symptomatic stage of Parkinson's disease based on the introduction of MPTP, similar to stages 1-2 on the Hoehn-Yar scale in patients with Parkinson's disease.

The authors of the present invention for the first time conducted a study of the stability of the expression of housekeeping in various tissues of the brain and peripheral blood of mice with modeling of the Parkinson-like phenotype based on the administration of MPTP and in the control group. The purpose of the study was to identify housekeeping that really have stable expression levels regardless of the development of the pathology under study in the body, as well as showing validation in specific experimental conditions, which leads to the possibility of blind transfer of housekeeping. selected for one organism to another, without concomitant careful checking.

To date, hundreds of reference genes are known both in the literature and in commercial panels. At the same time, the data of one expression study often do not reproduce the results of other similar studies; differences in the results obtained may be hidden in the choice and stability of housekeeping. A striking example is the comparison of the results for housekeeping Gapdh, which according to earlier data was listed as one of the most stable housekeeping among those considered, however, later, in 2020, the same group of scientists concluded that Gapdh has a highly variable expression in the process. reprogramming. On the contrary, in earlier works Rps18 was considered as one of the volatile housekeepings, however, in subsequent work, Rps18 was assigned to stable housekeepings.

Thus, the analysis of the available information on the housekeeping systems used does not allow us to identify housekeeping systems with stable expression and validity, however, due to the lack of the possibility to study all possible housekeeping systems. needed to narrow down the range of housekeeping that can be included in a study to determine stable expression and validity.

For this reason, the authors of the present invention screened the data in PubMed to select candidates for housekeeping. A MeSH search for the key terms "housekeeping gene" (filter: Humans) revealed 26,399 publications dating back to 2000. A keyword search for "housekeeping PCR" (filter: Humans) returned 880 results since 2000. A keyword search for "housekeeping real time PCR" (filter: Humans) returned 562 results since 2000. A keyword search for "housekeeping real time PCR brain" (filter: Humans) yielded 29 results since 2001. Further limitation on the scope of housekeeping out of these 29 results revealed 4 results directly related to the analysis of housekeeping in non-tumor specimens.

In addition, for the selection of potential housekeeping, the authors of the present invention used data from the analysis of reference genes in brain tissues, a neuronal line differentiated from bone mesenchymal stem cells, and human tissues. Using the data provided in the selected 7 studies, a list of potential housekeeping genes for further analysis was compiled, consisting of 542 genes.

Next, the authors of the present invention conducted another additional screening of publications for the query "gene expression Parkinson's disease rna" (filter: human) in the PubMed database and 715 publications were identified. The Materials and Methods section of 100 publications was randomly analyzed and a list of reference genes used in gene expression analyzes was compiled.

Further, the authors of the present invention compared the two generated lists, and for the subsequent study of expression stability and validity, genes included in both lists were selected. As a result of the analysis, housekeeping TBP, ASTV, RPL30, GAPDH, YWHAZ, PPIA, B2M, HPRT, GUSB, SARS, AARS, PSMD6, PSMD7, POLR2F, POLR2A, PSMA5 were selected.

Next, the authors of the present invention analyzed the selected housekeeping using the approaches established by the authors of the present invention. All potential housekeeping must meet certain conditions. First, there should be no pseudogenes of these genes in the genome. It is known that processed pseudogenes, lacking introns and being almost complete copies of mature mRNAs of the corresponding genes, account for 70% of all pseudogenes. In this regard, one cannot be sure that the selected primer systems will be absolutely specific to the target mRNA.

As a result of the bioinformatics analysis carried out by the authors of the present invention, pseudogenes were identified in such widely used housekeeping as ACTV, GUSB, PPIA, YWHAZ and GAPDH. For example, ACTV has 22 pseudogenes, while GAPDH has 47. Pseudogenes are able to be amplified together with cDNA derived from target RNA. On the one hand, treatment with DNases can serve as a solution to this problem; however, this greatly reduces the yield of isolated RNA. This method is of little use when the purpose of the study is to analyze changes in the levels of low-present transcripts. On the other hand, genomic DNA amplification can be avoided by selecting primers for inter-exon spikes, thus only the target cDNA derived from mature RNA lacking introns will be expressed. However, it is known that processed pseudogenes lacking introns are almost complete copies of mature mRNAs of the corresponding genes and account for 70% of all pseudogenes. In this regard, one cannot be sure that the selected primer systems will be absolutely specific to the target mRNA, and it is advisable not to use genes that have pseudogenes in the work.

The remaining housekeeping houses selected based on the results of the literature analysis underwent a bioinformatics analysis. It is important to note that for the PSMD7 (NM_002811.5) and HPRT1 (NM_000194.3) genes, pseudogenes were found in silico, however, there is currently no experimental confirmation of their existence, so the authors of the present invention suggested the possibility of selecting these genes for further analysis. , while weeding out such widely used housekeeping genes as ACTS, CUSB, PPIA, YWHAZ, RPL30, and GAPDH.

The second important approach established by the authors of the present invention was that a reference gene is a gene whose expression does not change under the conditions of the studied pathology, and is also approximately the same in the studied tissues. In this regard, the expression of all selected housekeeping was analyzed using BioGPS, and B2M housekeeping was excluded from further analysis based on the results of this analysis.

The inventors once again note that the literature review conducted prior to the main research work is due to the fact that hundreds of reference genes are known to date, both in the literature and in commercial panels. However, the disclosed information is the result of analytical work, while the study of the stability of expression and the validity of housekeeping for the analysis of gene expression in Parkinson's disease in mice, in particular in various tissues of the brain and peripheral blood of mice, was first carried out by the authors of the present invention. In addition, as already noted above, the authors of the present invention have identified their unique approach in identifying a number of housekeeping to be further investigated. As already stated above, housekeeping must satisfy several conditions. In particular, the expression levels of housekeeping should be stable regardless of the development of the studied pathology in the body. At the same time, from the knowledge available in this field of technology, the authors of the present invention included PSMD7 (NM_002811.5) and HPRT (NM_000194.3) housekeepings, for which in silica pseudogenes were found, into the series of housekeeping for research, which makes their inclusion in the series of housekeeping for research non-obvious based on the established approach to estimating housekeeping.

As a result, the authors of the present invention selected housekeeping POLR2F, POLR2A, PSMD6, PSMD7, HPRT, PSMA5, SARS, AARS, TBP and BCAT2, for which the authors of the present invention developed primer and probe systems using the TaqMan system to analyze the stability of the expression of selected housekeeping (table 1) and analyzed changes in their relative mRNA levels in brain and peripheral blood samples from mice.

As a result of a study pioneered by the present inventors with the aim of experimentally identifying housekeepings that show stable expression and validity in the analysis of gene expression in Parkinson's disease in mice, in particular in certain tissues of the brain and peripheral blood of mice, described in detail below in the "Examples" section ”, the authors of the present invention unexpectedly, based on previously available analytical data, identified a well-defined set of housekeeping Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, showing stable expression and validity in mice, in particular in certain tissues of the brain and peripheral blood mice.

At the same time, the TBP and BCAT2 genes unexpectedly showed an extremely low signal level for their detection, as a result of which they were excluded from the study.

At the same time, in an unexpected way, this set of housekeeping included PSMD7 and HPRT housekeeping, the inclusion of which in the study did not follow in an obvious way from the literature review conducted by the authors of the present invention, while Tbp and Polr2a housekeeping did not show stable expression in the course of the study, despite the absence in the study. provided for literature review of any information about the unstable expression of these housekeepings.

For the housekeeping set obtained, the authors of the present invention developed a set of primers and probes using the TaqMan system to determine the stable expression of the Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars housekeeping set in various tissues of the brain and peripheral blood of mice when analyzing gene expression in Parkinson's disease (Table 2).

As a result of the research work, the authors of the present invention managed not only to identify a strictly defined set of housekeeping Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, showing stable expression and validity in various tissues of the brain and peripheral blood of mice, but also to determine within the specified set of narrower sets of housekeepings, in particular, consisting of sin housekeepings, each of which shows stable expression and validity in certain tissues of the brain and peripheral blood of mice, which allows further analysis of changes in the expression of candidate genes for each specific cell line.

The authors of the present invention investigated the stability of the expression of a selected set of housekeeping in various tissues of the brain and peripheral blood of mice, and unexpectedly found that:

- in the brain as a whole, housekeeping Aars, Psmd7 and Psmd6 are most stably expressed,

- housekeeping Polr2f, Aars and Bcat2 are most stably expressed in the cortex,

- housekeeping Psmd6, Aars and Polr2f are most stably expressed in the cerebellum,

- housekeeping Psmd7, Aars and Sars are most stably expressed in the striatum,

- housekeeping Psmd7, Aars and Bcat2 are most stably expressed in substantia nigra,

- housekeeping Polr2f, Hprt and Sars are most stably expressed in peripheral blood.

In particular, the Aars gene is the most stably expressed housekeeping in the brain as a whole. The Psmd7 gene is the most stably expressed housekeeping in the striatum and substantia nigra. In the cortex and peripheral blood, the most stably expressed housekeeping is the Polr2f gene, and in the cerebellum, the Psmd6 gene.

In addition, during the study conducted by the authors of the present invention, they unexpectedly found that the well-defined set of housekeeping developed as a result of this study shows stable expression regardless of the presence of a pathological process.

The authors of the present invention were able to obtain these results due to the fact that, in the framework of their research work, it was initially concluded that the importance of stable expression of housekeeping, regardless of the presence of a pathological process, was important.

Thus, the authors of the present invention obtained a strictly defined set of housekeeping Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, which makes it possible to study the pathological process associated with the pathogenesis of Parkinson's disease in mice.

As a result of a set of housekeeping Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars unexpectedly identified during the study, showing stable expression in the analysis of gene expression in Parkinson's disease in various tissues of the brain and peripheral blood of mice, as well as through the developed by the authors of the present invention set of primers and probes to determine the stable expression of the specified set of housekeeping in various tissues of the brain and peripheral blood of mice, the authors of the present invention also developed a method for analyzing gene expression in Parkinson's disease, involving the use of the specified set of housekeeping in various tissues of the brain and peripheral blood of mice with using the specified set of primers and probes.

The set of primers and probes developed by the authors of the present invention, as well as the method developed by the authors of the present invention for analyzing gene expression in Parkinson's disease, provide an effective analysis of gene expression in Parkinson's disease due to the use of an unexpected, strictly defined set of Aars, Bcat2, Hprt, Polr2f, Psmd6 housekeeping identified during the study. , Psmd7, Sars, showing stable expression and validity in mice, as well as narrower housekeeping sets within this set for analysis of gene expression in Parkinson's disease in certain tissues of the brain and peripheral blood of mice.

Examples

List of abbreviations and symbols

HKG - housekeeping

Comprehensive score - complex score

Gene rank - gene rank

MPTP-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

RT-qPCR - quantitative real-time PCR.

Example 1 Determination of a Set of Housekeeping Showing Stable Expression and Validity in Mice for Gene Expression Analysis in Parkinson's Disease

Twenty C57BL/6 mice were purchased from a special licensed pathogen-free nursery, Institute of Bioorganic Chemistry. Shemyakin and Ovchinnikov RAS. Eight-week-old male mice weighing 22-25 g were randomly divided for analysis into two groups: control and experimental. Each group of mice consisted of 10 individuals. Mice were placed 3-4 animals per cage under standard conditions with free access to food and water with a light:dark regimen of 12:12 hours.

Modeling the parkinson-like phenotype using MPTP

This study used a model of the early symptomatic stage of Parkinson's disease in mice. Experimental work with laboratory animals was carried out in accordance with the Animal Care Guidelines approved by the Ethics Committee of the Institute of Molecular Genetics, National Research Center "Kurchatov Institute" (Moscow, Russia), protocol 3/21 dated February 17, 2021. After decapitation, each animal was taken for research samples of 5.5±0.35 mg of striatum, cortex and cerebellum, 1.4±0.22 mg of substantia nigra and 477.8±80 µl of peripheral blood.

Every effort has been made to minimize potential suffering and discomfort to the animals.

Expression analysis

Isolation of RNA

Isolation of total RNA from brain tissues was performed using RNAeasy Mini Kit (Qiagen, Germany) and Quick-RNA MiniPrep Kit™ (Zymo Research Corp., USA).

Isolation of total RNA from 250 µl of each sample of human and mouse peripheral blood was performed using the MiniPrep Kit for whole RNA (Zymo Research Corp., USA).

The concentration of isolated RNA from the samples was measured using the Quant-iT RNA BR Assay Kit and a Qubit 3.0 fluorimeter (Invitrogen, USA). RNA quality was monitored using an Experion automated electrophoresis system (Bio-Rad Laboratories). The RNA quality index was above 8.5 in all samples.

All procedures were performed according to the manufacturer's recommendations.

Analysis of changes in relative mRNA levels

Gene expression analysis was performed using real-time PCR with TaqMan probes. All samples were processed as evenly as possible to minimize any random bias. Single-stranded DNA was synthesized using 120 ng of total RNA, 100 ng of Escherichia coli tRNA as a carrier, specific primers, and the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fischer Scientific, USA) according to the manufacturer's recommendations. Each reaction was carried out in three repetitions.

Primers and probes for real-time PCR were selected using the Beacon Designer 7.0.2 program and nucleotide sequences of human, mouse, and rat genes. The sequences of the gene-specific primers and probes are listed in Table 1 above.

cDNA obtained in the reverse transcription reaction was used as a template for quantitative PCR. It was diluted 50 times with an aqueous solution of tRNA from Escherichia coli (0.02 ng/µl). PCR was performed using the StepOnePlus™ system (Applied Biosystems, USA), QuantStudio™ 3 system (Thermo Fisher Scientific. USA) and PCR reagents (Syntol, Russia). Temperature nicks for mouse and human samples were performed as follows: 60 s at 50°C, 40 cycles of 15 s at 95°C and 40 s at 61°C; 30 s at 25°C. The thermal cycle for samples from rats was performed as follows: 600 s at 95°C and 45 cycles: 5 s at 95°C, and then 10 s at 60°C. The reaction was repeated three times for each cDNA to correct for differences in sample quality and reverse transcription efficiency.

Statistical analysis

Primers and probes for analysis were selected using Beacon Designer 7.0 (Premier Biosoft International, USA) and corresponding sequences from the NCBI database. The specificity of primers and probes was checked using Primer3, Primer-BLAST. Reference gene expression was assessed using threshold amplification cycle values and RefFinder software.

The RefFinder software calculated the reference gene scores using the geNorm, Normfinder, BestKeeper algorithms and the ΔΔCt comparison method.

Based on these scores, the software assigned each gene an appropriate score and calculated the geometric mean of their weights for an overall final ranking. The program selects the lowest score among the ranks assigned by each algorithm separately, and then assigns the final overall score. The closer the final score is to one, the more stable housekeeping is considered. The results are presented in table 3.

results

To identify the most stable HKG under Parkinson's disease, HKG stability was analyzed separately in a group of control mice (Table 3A) and separately in a group of mice with an MPTP-induced Parkinson's disease model (Table 3B). Stability was assessed based on the calculation of a complex score for each gene in each tissue. In each specific case (tissue and presence/absence of pathology), the most stable is HKG, which has the minimum complex score. So. for example, the most optimal and stable HKG in the brain as a whole, as well as in three other brain tissues (cerebellum, striatum and substantia nigra) in the control group of mice is Psmd7, however, in the cortex this gene occupies 2nd place, and in the first place Aars1 is located. At the same time, in the group of experimental animals with modeling of Parkinson's disease in the brain, Psmd6 is generally the most stable, however, in the cortex and striatum it occupies only the 4th place, and in the cerebellum and substantia nigra - the 3rd place.

The results show that the stability of the expression of the studied housekeeping differs depending on the presence or absence of pathological processes. However, the task of the study was to determine housekeeping, showing stable expression regardless of the presence of pathology. For this reason, it became necessary to conduct further summary statistical analysis in order to determine the total complex score, the value of which will allow us to identify housekeeping showing stable expression regardless of the presence of pathology.

Determination of the total complex score

Based on the obtained results, that stability in different tissues and in the presence and absence of pathology differs, when carrying out work on expression analysis, it is necessary to use housekeeping, whose expression is most stable in a particular tissue both in the presence and in the absence of pathology. Based on this, the final rank of housekeeping was calculated based on the calculation of the total complex score (Table 4). The total composite score for each housekeeping in each tissue was summed from the composite scores obtained from the analysis of the control group of mice (table 3A) and mice with a simulation of parkinson-like phenotype (table 3B).

So, for example, in the brain as a whole, regardless of the presence or absence of pathology, Aars1 is the most stably expressed and takes the first place (Gene rank=1) with a total complex score of 4.21-2.21+2.20, where 2.21 is the value of the complex score from column "whole brain" of table 3A, and 2.20 is the value of the composite score from the column "whole brain" table 1B. Thus, the gene with the lowest value is considered as the most stably expressed and takes the first place in the ranking (Gene rank=1).

results

As a result of the study, a set of housekeepings was determined, consisting of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, which show stable expression and validity in mice, regardless of the presence of pathology. The identification of this set of housekeeping is an unexpected result of the study, since the housekeeping for the study were equally selected according to the approach established by the authors of the present invention, with the exception of the Psmd7 housekeeping, which was included by the authors of the present invention in the study, despite the fact that pseudogenes in silica. At the same time, in the literature submitted for review, there was no information on unstable expression of housekeeping, which was not included in the set of housekeeping identified according to the study, showing stable expression and validity in mice.

At the same time, a study for brain tissues in general, as well as for the cortex, cerebellum, striatum and substantia nigra, and for peripheral blood revealed that:

- in the brain as a whole, housekeeping Aars, Psmd7 and Psmd6 are most stably expressed,

- housekeeping Polr2f, Aars and Bcat2 are most stably expressed in the cortex,

- housekeeping Psmd6, Aars and Polr2f are most stably expressed in the cerebellum,

- housekeeping Psmd7, Aars and Sars are most stably expressed in the striatum,

- housekeeping Psmd7, Aars and Bcat2 are most stably expressed in substantia nigra,

- housekeeping Polr2f, Hprt and Sars are most stably expressed in peripheral blood.

These tissue-specific brain and peripheral blood results are based on the widely accepted approach in the art that when analyzing changes in gene expression, at least two housekeeping should be taken into account and genes located on the 1st, 2nd and maximum on 3rd place based on the resulting gene rank.

In general, based on the obtained data, Aars housekeeping has the most stable expression among all studied housekeeping in the brain. The same gene ranks second in terms of expression stability in various parts of the brain. Occupying the 2nd place in the entire brain, Psmd7 was most stably expressed in the striatum and substantia nigra. Polr2f housekeeping was most stably expressed in the cortex and Psmd6 housekeeping in the cerebellum. It is noteworthy that the first of them was also most stably expressed in the peripheral blood of mice.

Thus, the authors of the present invention were able to identify a set of housekeeping consisting of at least 3 housekeeping from the group consisting of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, for use in the analysis of gene expression in Parkinson's disease, where the specified set housekeeping shows stable expression in various tissues of the brain and peripheral blood of mice and shows validity, and develop a set of primers and probes to determine the stable expression of a housekeeping set consisting of at least 3 housekeeping from the group consisting of Aars, Bcat2, Hprt, Polr2f , Psmd6, Psmd7, Sars, in various tissues of the brain and peripheral blood of mice and a method for analyzing gene expression in Parkinson's disease, using a set of housekeeping consisting of at least 3 housekeeping from the group consisting of Aars, Bcar2, Hprt, Polr2f, Psmd6, Psmd7, Sars, in various tissues of the brain and peripheral blood of mice using the developed set of primers and probes, where the developed set of primers and probes and the expression analysis method provide an effective analysis of gene expression in Parkinson's disease.

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SEQUENCE LIST

<110> Federal State Budgetary Institution Institute of Molecular

genetics of the National Research Center "Kurchatov Institute"

<120> housekeeping kit for analysis of gene expression in Parkinson's disease

in various tissues of the BRAIN and peripheral blood of mice and

its application

Aars probe

5'-FAM-ACCCCACTCTGCTCTTCGCCAACG-BHQ1-3'

Aars-direct primer

5'-TCCACTCTTCCGCCACCAT-3'

Aars-reverse primer

5'-CTGCTCAGCTTCGCCATAGG-3'

Bcat2-Probe

5'-FAM-CGGATACACTCCAACAGCTCCTGCTTGT-BHQ1-3'

Bcat2 forward primer

5'-TCAACATGGACAGGATGCTACG-3'

Bcat2 reverse primer

5'-CCAGTCTTTGTCTACTTCAATGAGC-3'

Hprt probe

5’- FAM- CCTGTATCCAACACTTCGAGAGGTCCTT- BHQ1-3’

Hprt-direct primer

5'-ACAGCCCCAAAATGGTTAAGGT-3'

Hprt-reverse primer

5'-CCAACAACAAACTTGTCTGGAATTTC-3'

Polr2f-probe

5'- VIC-CCTCAGCATTTTCCAAGTCGTCAAGTCC-BHQ2-3'

Polr2f-forward primer

5'- TGTCAGACAACGAGGACAATTTCG-3'

Polr2f reverse primer

5'- TCGCTCACCAGATGGGAGAATC-3'

psmd6-probe

5'- FAM-AATCGTGGAGACCAACAGACCTGATAGCAA- BHQ1-3'

Psmd6 - forward primer

5'-GGTGTGGGTGTGGACTTCATT-3'

Psmd6 reverse primer

5'-CTCCTTTCTTGATGGTTTCTTGATACTG-3'

psmd7-probe

5'-FAM-AGTCCTAGGTCCTTTGGCTTCACGTCGA-BHQ1-3'

Psmd7-forward primer

5'-CTGCACAAGAATGATATCGCCATC-3'

Psmd7 reverse primer

5'-CTCCACTGAGATGTAGGCTTCG-3'

Sars probe

5’- FAM- CGTTCTACTTTGTTGTCTGCGTCCTCATCA- BHQ1-3’

Sars forward primer

5'-GCGAGATTGGGAACCTTCTG-3'

Sars reverse primer

5'-ATGGGAATACTTCTTCCTGACTGTA-3'

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Claims (12)

1. A set of primers and probes to determine the stable expression of a housekeeping set consisting of at least 3 housekeepings from the group consisting of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, in brain tissues and peripheral blood of mice in the analysis of expression genes in Parkinson's disease, where the set of primers and probes is:

2. A method for analyzing gene expression in Parkinson's disease, involving the isolation of RNA from tissue samples of the brain and peripheral blood and the use of a set of primers and probes according to claim 1 to determine the stability of the expression of a set of housekeeping, consisting of at least 3 housekeeping selected from the group, consisting of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars, in brain tissues and peripheral blood of mice. 3. The method according to claim 2, wherein the housekeeping set consists of Aars, Bcat2, Hprt, Polr2f, Psmd6, Psmd7, Sars and is stably expressed in brain tissues and peripheral blood of mice. 4. The method of claim 2, wherein the brain tissue is the brain as a whole. 5. The method of claim 2, wherein the brain tissues are selected from the group consisting of cortex, cerebellum, striatum, and substantia nigra. 6. The method according to claim 2 or 4, wherein the set of housekeeping consists of housekeeping Aars, Psmd7 and Psmd6 and is stably expressed in the brain as a whole. 7. The method according to claim 2 or 5, wherein the housekeeping set consists of Polr2f, Aars and Bcat2 housekeeping and is stably expressed in the cortex. 8. The method according to claim 2 or 5, wherein the housekeeping set consists of Psmd6, Aars and Polr2f housekeeping and is stably expressed in the cerebellum. 9. The method of claim 2 or 5, wherein the housekeeping set consists of Psmd7, Aars and Sars housekeeping and is stably expressed in the striatum. 10. The method of claim 2 or 5, wherein the housekeeping set consists of Psmd7, Aars and Bcat2 housekeeping and is stably expressed in the substantia nigra. 11. The method of claim 2, wherein the housekeeping set consists of Polr2f, Hprt and Sars housekeeping and is stably expressed in peripheral blood.

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