Group of plasma non-coding RNA, primer group for detecting expression level of plasma non-coding RNA and colorectal cancer (CRC) detection kit

A detection kit and technology for colorectal cancer, which are applied in the fields of genetic engineering and clinical medicine, can solve the problems that affect the diagnostic accuracy and positive rate of colorectal tumors, are not suitable for large-scale clinical use, and have low expression of non-coding RNAs. Achieve stable expression, easy detection, and accurate quantification

Active Publication Date: 2021-01-05
JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing detection of non-coding RNA markers in peripheral blood has false positive interference, and due to the low expression of non-coding RNAs, the requirements for detection are high, so it is not suitable for large-scale clinical application. The distinction between rectal cancer (CRC) and colorectal adenoma (CRA) indirectly affects its diagnostic accuracy and positive rate in colorectal neoplasms
At present, there are no research reports on non-coding RNA in peripheral blood of patients with colorectal cancer

Method used

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  • Group of plasma non-coding RNA, primer group for detecting expression level of plasma non-coding RNA and colorectal cancer (CRC) detection kit
  • Group of plasma non-coding RNA, primer group for detecting expression level of plasma non-coding RNA and colorectal cancer (CRC) detection kit
  • Group of plasma non-coding RNA, primer group for detecting expression level of plasma non-coding RNA and colorectal cancer (CRC) detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Research Object Selection and Grouping Basis

[0025] The purpose of this embodiment is to establish a unified specimen bank and database, collect standard blood samples with standard operating procedures, and systematically collect complete demographic and clinical data. In addition, in the same way, blood samples were taken from blood donors who were diagnosed with colorectal polyps and excluded from the diagnosis of colorectal cancer in the gastroenterology department during the same period, and blood samples were taken from those who underwent routine physical examination. All experiments were approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. This study was conducted in accordance with the guidelines set by the Declaration of Helsinki.

[0026] Specific sample classification criteria are as follows:

[0027] Group A: Healthy control group (n=600, 3 persons for microarray screening, 597 persons for first-s...

Embodiment 2

[0043] Example 2 Validation of Candidate Plasma miRNA Biomarkers

[0044] 1. Total RNA extraction and cDNA sample preparation:

[0045] The purpose of this example is to extract the RNA in the sample and prepare the cDNA required for subsequent experiments. The method used in this example is a conventional molecular biology method, and the reagents used are all commercially available (commodities / reagents). The steps of the specific example are as follows :

[0046] a) Take 100 microliters of plasma and add 5 milliliters of fresh heparin anticoagulant blood, centrifuge at 3000 rpm for 5 minutes in a centrifuge, and absorb the supernatant;

[0047] b) Add 900 microliters of Trizol reagent, oscillate and mix well, centrifuge at 12000 rpm for 15 minutes at 4 degrees, take the supernatant, and discard the waste liquid in the lower layer;

[0048] c) Add 1.5 times the volume of supernatant absolute ethanol, shake and mix, transfer to a spin column, centrifuge at 12,000 rpm for 15...

Embodiment 3

[0071] Example 3 Validation of Candidate Plasma lncRNA Biomarkers

[0072] The purpose of verification in this example is to verify the expression levels of the six lncRNA biomarkers screened in Example 1. Extraction of total RNA was as described in Example 2 above. The specific steps of cDNA required for reverse transcription follow-up experiments are as follows: take the pretreated plasma RNA, and obtain cDNA samples through RNA reverse transcription reaction. Prepare the required reaction system according to the instructions, as shown in Table 5 below:

[0073] table 5

[0074]

[0075] After the above system was mixed, it was centrifuged briefly, and the RT reaction program was: 42°C for 60 minutes, 70°C for 10 minutes.

[0076] 2. Detect the expression of plasma miRNA biomarkers obtained in Example 1, and verify the expression of plasma lncRNAs by RT-PCR method:

[0077] Prepare the qPCR reaction system on ice, as shown in Table 6 below:

[0078] Table 6

[0079]...

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Abstract

The invention provides a group of plasma non-coding RNA, a primer group for detecting the expression level of the plasma non-coding RNA and a colorectal cancer (CRC) detection kit. The plasma non-coding RNA is composed of plasma non-coding RNA of which the base sequence is shown as SEQ ID NO.1-SEQ ID NO.6 in sequence, and the primer group sequence for detecting the plasma non-coding RNA group is shown as SEQ ID NO.7-SEQ ID NO.18 in sequence. The primer group can be applied to preparation of the CRC detection kit; The plasma miRNA and lnc RNAs biomarkers are jointly applied for the first time,compared with a single biomarker, the sensitivity and specificity of colorectal tumor diagnosis are improved, and great clinical significance and application value are achieved for early diagnosis ofCRC and postoperative monitoring of tumor recurrence.

Description

technical field [0001] The invention belongs to the field of genetic engineering and clinical medicine, and relates to the preparation of a group of non-coding RNA in plasma related to the occurrence and development of colorectal cancer and its primer set in the preparation of a detection kit for early screening of colorectal tumors and postoperative monitoring of colorectal tumor recurrence in the application. Background technique [0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract, with high morbidity, high mortality, and poor prognosis. According to statistics, the morbidity and mortality of colorectal cancer rank third and second among cancers, respectively. In recent years, with the continuous development of medical science and the continuous improvement of diagnosis and treatment technology, a variety of treatment methods including neoadjuvant, surgery, and immunotherapy have made great progress. Many patients with colore...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/113C12N15/11
CPCC12Q1/6886C12Q2600/158C12Q2600/166C12Q2600/178Y02A50/30
Inventor 孙跃明唐俊伟李杰陈冉冉封益飞张冬生
Owner JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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