NEW GENERATION PROGNOSTIC QPCR PANEL DIAGNOSTIC KIT FOR BREAST CANCER DIAGNOSIS.

Abstract

The invention relates to a new generation prognostic qPCR panel diagnostic kit to be used in the diagnosis of breast cancer, created with an RNA isolation unit (1) in which the mRNA levels of the genes KIF11, CDK1, CCNA2, Ki67 (MKI67), BUB1, ADAMTS5, ADH1A, TIMP4, BAG1 are converted to cDNA using the OneScript Plus cDNA Synthesis Kit protocol, a cDNA synthesis unit (2), a SYBR Green based qPCR unit (3) for calculating the gene expression levels of these genes converted to cDNA according to the mRNA levels of the housekeeping gene GAPDH, and a data analysis device (4) that provides information about breast cancer diagnosis and prognosis by evaluating the data from the SYBR Green based qPCR unit (3).

Description

-1- TARIFF qPCR, a next-generation prognostic test, will be used in the diagnosis of breast cancer. PANEL DIAGNOSTIC KIT Technical Area The invention relates to the qPCR panel diagnostic kit used in the diagnosis of breast cancer. Specifically, Designed for 9 genes associated with breast cancer and 1 housekeeping gene. Real-time PCR using unique primers and SYBR Green technology. Properly formulated, fast with results in 70 minutes, at a single temperature of 56°C and in a single step. By enabling all genes to work simultaneously on the plate, user 10 friendly, able to yield results in breast cancer diagnosis in different breast cancer cell lines This relates to the new generation prognostic qPCR panel diagnostic kit that will be used. Description of the State of the Art and Known Technical Problems Breast cancer is one of the most commonly diagnosed types of cancer worldwide. Many patients die each year due to delayed diagnosis. Prognosis, or prediction, is a in terms of an estimate of the disease's course and whether there is a chance of recovery. It is a medical term used. Breast cancer is a leading cause of high mortality among women. This is one of the reasons, and many breast cancer patients suffer from delayed diagnosis and treatment. They lose their lives due to this. In addition, 5 of the early-stage breast cancer patients The annual survival rate is over 90%; however, this can change when metastases occur. The rate drops dramatically to approximately 27%. Therefore, breast 25 Early diagnosis of cancer is critical and important. Breast cancer is one of the leading causes of death in women worldwide. It is the most common type of cancer diagnosed in women worldwide. Every year, many breast... Cancer patients are dying due to delayed diagnosis and treatment. ER, PR, Ki-67 and HER2 30 According to the (ERBB2) status, MK, luminal A, is characterized by significant heterogeneity. There are four main types: luminal B, HER2-positive and triple-negative breast cancer (TNBC). They can be divided into subtypes. Statistically, 95% of breast cancers are adenocarcinomas. -2- They are classified, and 55% of all patients may progress to invasive stages; however, the initial If diagnosed in the early stages, approximately 70-80% of cases can be successfully treated. Furthermore, the 5-year survival rate for patients with early-stage breast cancer is over 90%. It is above this level, whereas when metastasis occurs, this rate dramatically increases to approximately 27%. It is falling in this way. Given the variability in the clinical course of the disease, tumors in breast cancer Identifying markers that can predict behavior is extremely important. Furthermore, Determining tumor markers is a useful tool in the clinical management of cancer patients. They function as tools. These markers are used in diagnostic processes and staging. in the stages of evaluating the response to treatment, recurrence and distant metastases while helping in diagnosis and prognosis, new treatment methods It also contributes to their development. Therefore, new molecular biomarkers and Seeking therapeutic targets improves the survival and prognosis of breast cancer patients. It is extremely important for improvement. In Chinese application number CN113481296 (A), early detection of breast cancer risk A diagnostic kit for evaluation has been described. The diagnostic kit includes a standard substance, long-chain... a non-coding RNA (Ribonucleic Acid), two specific gene primers, and a reference gene primer 20 It includes and the targeted detection genes and reference genes are LINC00504 (GeneID: 201853), RFX5 (GeneID: 5993), VEGFA (GeneID: 7422) and beta-actin (GeneID: 60); currently, methods such as SNP (Single Nucleotide Polymorphism) regions and the like. It is used to analyze the initial risk assessment of breast cancer. Its applicability is weak, it lacks effective sensitivity and specificity, and the method's actual detection is 25. and cannot be used for the application. This will make it easier to diagnose breast cancer and provide information about its prognosis. Work is underway on new technical developments to meet this need. In this context, the development of new techniques has become essential. 30 -3- Explanation of the Purposes of the Invention and the Problems It Is Intended to Solve Given the current state of the art, the aim of the invention is to... breast New generation prognostic qPCR panel diagnostic kit to be used in cancer diagnosis 5 It is the development of. Another aim of the invention is to make it easier to diagnose breast cancer and a fast, economical, user-friendly qPCR meme that will provide information about the prognosis The goal is to develop a cancer diagnostic kit. 10 Another aim of the invention is to identify the most current potential findings related to breast cancer. This involves selecting prognostic markers and designing primary care plans. Another objective of the invention is to optimize SYBR Green 15 with the primers produced. MCF-7 and MDA-MB-231 cells, representing breast cancer, were used in a qPCR reaction based on the acronym qPCR. The goal is to develop a diagnostic kit that can be tested on bloodstreams. Another aim of the invention is to identify 9 genes and 1 gene associated with breast cancer. SYBR Green 20 is unique because it has primers designed for the housekeeping gene. Suitable for real-time PCR technique using advanced technology, it provides results in 70 minutes. Fast, at a single temperature of 56°C and on a single plate, all genes simultaneously. It is user-friendly as it allows for research on different breast cancer cell lines. A new generation prognostic qPCR panel that yields conclusive results for breast cancer diagnosis. It is the development of a diagnostic kit. 25 In order to achieve the objectives of the invention, breast cancer, which offers many advantages... A new generation prognostic qPCR panel diagnostic kit has been developed for use in diagnosis. Explaining the Figures 30 Figure 1: A drawing illustrating a representative application of the invention. -4- Reference Numbers 1 mRNA isolation unit, 4 data analyzers 2 cDNA synthesis units 3 SYBR Green based qPCR unit Detailed Description of the Invention and Alternative Designs The invention relates to the following: KIF11, CDK1, CCNA2, Ki67 (MKI67), BUB1, ADAMTS5, ADH1A, TIMP4, 5 mRNA levels of BAG1 genes were determined using the OneScript Plus cDNA Synthesis Kit protocol. RNA isolation unit (1), where it is converted to cDNA, cDNA synthesis unit (2), made These genes, which are translated into cDNA, have gene expression levels similar to those of the housekeeping gene GAPDH. SYBR Green-based qPCR for calculating gene mRNA levels. unit (3), SYBR Green based qPCR unit (3) data evaluated and breast 10 created with a data analysis device (4) that provides information about cancer diagnosis and prognosis With the new generation prognostic qPCR panel diagnostic kit to be used in the diagnosis of breast cancer It is related. 15 designed for 9 genes associated with breast cancer and 1 housekeeping gene. Real-time PCR using unique primers and SYBR Green technology. Properly formulated, fast with results in 70 minutes, at a single temperature of 56°C and in a single step. by enabling all genes to work simultaneously on the plate, the user friendly, able to yield results in breast cancer diagnosis in different breast cancer cell lines A new generation prognostic qPCR panel diagnostic kit has been developed for use. 20 The invention will make it easier to diagnose breast cancer and improve its prognosis. It will provide information, is fast, economical, user-friendly, and is a domestically produced qPCR breast cancer scanner. A diagnostic kit has been developed. Nine genes were selected for this purpose: KIF11, CDK1, CCNA2, The primary mRNA levels of the Ki67 (MKI67), BUB1, ADAMTS5, ADH1A, TIMP4, and BAG1 genes are 25%. These levels are converted to cDNA using the OneScript Plus cDNA Synthesis Kit protocol. mRNA isolation unit (1) is used for this. These genes, which are converted to cDNA, are used for gene expression. -5- levels according to mRNA levels of the GAPDH gene, which is a housekeeping gene For calculation, it is processed in the cDNA synthesis unit (2). These genes, which were converted to cDNA in the SYBR Green based qPCR unit (3), Expression levels are 5 based on mRNA levels of the GAPDH gene, which is a housekeeping gene. The calculation is transferred to the data analysis device (4) and here the diagnosis is made together with the predictions. The result is generated. This creates a new method to be used in the diagnosis of breast cancer. Thanks to the next-generation prognostic qPCR panel diagnostic kit, predictions can be made more quickly and easily. Diagnosis can be made using the ct values ​​obtained from the qPCR results. Calculations are based on the percentage changes in gene expression tested in the cell line, and data analysis is used. 10 By evaluating the data in the device (4), diagnosis and prediction can be made. This will make it easier to diagnose breast cancer and provide information about its prognosis. To provide a fast, economical, and user-friendly diagnostic kit, KIF11, CDK1, mRNA levels of CCNA2, Ki67 (MKI67), BUB1, ADAMTS5, ADH1A, TIMP4, and BAG1 genes are 15%. RNA levels were converted to cDNA using the OneScript Plus cDNA Synthesis Kit protocol. isolation unit (1), cDNA synthesis unit (2), this is converted into cDNA genes, gene expression levels of the housekeeping gene GAPDH gene mRNA SYBR Green based qPCR unit (3) for calculation according to levels, SYBR By evaluating the data of the Green-based qPCR unit (3), breast cancer diagnosis and 20 It is characterized by containing a data analysis device (4) that provides information about the prognosis. is being done. Calculations related to ct values ​​obtained from qPCR results are performed in the cell line. Data that enables diagnosis and prediction based on the percentage changes in the expression of the tested genes. 25 The analysis device (4) has been created. Real-time PCR reaction, forward and reverse primers specific to the synthesized gene. The GAPDH gene, as the Housekeeping gene, was measured using fluorescence emission. Gene expression levels in samples were determined using housekeeping genes reference 30. A SYBR Green based qPCR unit (3) was created by generating the data. -6- RNA isolation unit (1) where RNA samples are taken and stored at -80°C It was created. All processes for carrying out DNA (cDNA) synthesis are performed on ice. cDNA synthesis unit (2) was created in the environment in which it was made.

Claims

-7- REQUESTS 1. Invention: A new generation prognostic qPCR panel for the diagnosis of breast cancer. It is a diagnostic kit whose feature is that it will facilitate the diagnosis of breast cancer and A fast, economical, user-friendly diagnostic kit that will provide information about the prognosis. 5 to create, - KIF11, CDK1, CCNA2, Ki67 (MKI67), BUB1, ADAMTS5, ADH1A, mRNA levels of TIMP4 and BAG1 genes were obtained using OneScript Plus cDNA. RNA isolation unit where RNA is converted to cDNA using the Synthesis Kit protocol. (1), cDNA synthesis unit (2), 10 - The gene expression levels of these genes, which were converted into cDNA. based on mRNA levels of the GAPDH gene, which is a housekeeping gene SYBR Green based qPCR unit for calculation (3), - By evaluating the data of the SYBR Green based qPCR unit (3) Data analysis device 15 that provides information about breast cancer diagnosis and prognosis. (4) is characterized by its inclusion.

2. Next-generation prognostic device for use in breast cancer diagnosis, in accordance with Claim 1. qPCR panel is a diagnostic kit and its features include: - Calculations related to ct values ​​obtained from qPCR results, cell 20 Diagnosis and prediction using percentage changes in gene expression tested on the line. It includes a data analysis device (4) that enables placement.

3. Next-generation prognostic devices for breast cancer diagnosis, in accordance with Claim 1. qPCR panel diagnostic kit, its feature is 25 - Real-Time PCR reaction uses forward and reverse PCR reactions specific to the synthesized gene. Housekeeping gene by using primers and measuring fluorescence Gene expression levels in samples were determined using the GAPDH gene. SYBR Green creates data based on housekeeping genes. It contains a qPCR unit (3) based on 30. -8- 4. Next-generation prognostic devices for breast cancer diagnosis, in accordance with Claim 1. qPCR panel is a diagnostic kit and its features include: - RNA isolation where RNA samples were collected and stored at -80°C. It contains unit (1).

5. Next-generation prognostic devices for breast cancer diagnosis, in accordance with Claim 1. qPCR panel is a diagnostic kit and its features include: - All processes for DNA (cDNA) synthesis are carried out in an ice environment. It contains the cDNA synthesis unit (2) from which it is made.