Internal reference substance for detecting bladder cancer serum miRNA and its detection primers and use

A technology for detecting primers and bladder cancer. It is applied in the directions of DNA/RNA fragments, recombinant DNA technology, and microbial measurement/inspection. It can solve problems such as lack of internal reference molecules, inconsistent internal references, and restrictions on the transformation of research results. Application prospects, elimination of expression level differences, stable and reliable detection efficiency

Active Publication Date: 2014-11-05
SHANDONG UNIV QILU HOSPITAL
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, in the study of serum miRNA in bladder cancer, there is still a lack of recognized and relatively stable internal reference molecules. In the past, researchers mainly selected internal reference molecules based on their own experience or references. The internal reference molecules selected in different experiments are usually inconsistent, thus restricting The transformation of research results and the comparison of research results between different laboratories

Method used

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  • Internal reference substance for detecting bladder cancer serum miRNA and its detection primers and use
  • Internal reference substance for detecting bladder cancer serum miRNA and its detection primers and use
  • Internal reference substance for detecting bladder cancer serum miRNA and its detection primers and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Collection of Specimen and Data Arrangement

[0044] The inventors of the present invention collected serum samples from 250 bladder cancer patients from Qilu Hospital of Shandong University from 2011 to 2013, and collected serum from 158 control subjects during the same period, which were used as experimental samples for Solexa sequencing and subsequent qRT-PCR screening and verification. .

[0045] (1) 250 patients with bladder cancer diagnosed by pathology had not undergone surgery, radiotherapy and chemotherapy before blood collection.

[0046] (2) A total of 158 healthy male and female controls were included.

[0047] The demographic data and clinicopathological data of the above subjects were systematically collected.

Embodiment 2

[0048] Example 2 Solexa sequencing of serum miRNA

[0049] The samples selected in the sequencing stage included 10 invasive bladder cancers, 10 non-invasive bladder cancers, and 10 controls. The three groups of samples were sequenced by Solexa to obtain relevant results (the kits were purchased from ABI). The specific steps are:

[0050] (1) Take 15ml of serum from each group of samples, add an equal volume of Trizol reagent and mix thoroughly;

[0051] (2) Place at room temperature for 30 minutes, then add chloroform at a volume ratio of 0.2 ml of chloroform per 1 ml of Trizol reagent, shake vigorously for 10 s, 20 minutes at room temperature, 12,000 g, 4 °C, and centrifuge for 15 minutes;

[0052] (3) Carefully transfer the supernatant to a new centrifuge tube, and use the 3-step phenol / chloroform method to remove the protein;

[0053] (4) Transfer the aqueous phase to a new centrifuge tube, then add isopropanol at a volume ratio of 0.5 ml of isopropanol per 1 ml of Trizo...

Embodiment 3

[0062] Example 3 qRT-PCR screening and verification of serum miRNA

[0063] According to Solexa sequencing results, miRNA molecules meeting the following criteria were selected for further screening using qRT-PCR technology: a) The expression copy number was greater than 50 in invasive bladder cancer, non-invasive bladder cancer and the control group; b) In the three groups All were stably expressed, and there was no significant difference between the three groups (p≥0.05). According to the above criteria, a total of 10 miRNA molecules that meet the requirements (including hsa-miR-193a-5p, hsa-miR-191-5p, hsa-miR-16-5p, hsa-miR-10a-5p, hsa- miR-345-5p, hsa-miR-143-3p, hsa-miR-140-3p, hsa-miR-502-3p, let-7d-3p, hsa-miR-141-3p). In addition, since U6 is often used as an internal reference molecule for tissue miRNA detection, in order to verify whether it can be used as an internal reference in serum, U6 was also included in the screening as a candidate molecule. The above 10 m...

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Abstract

The invention discloses an internal reference substance for detecting bladder cancer serum miRNA. The internal reference substance is single hsa-miR-193a-5p or a mixture of hsa-miR-193a-5p and hsa-miR-16-5p. The invention also discloses detection primers of the internal reference substance. The detection primers comprises primers for detecting hsa-miR-193a-5p (shown in the formulas of SEQ ID NO: 1-3) or / and hsa-miR-16-5p (shown in the formulas of SEQ ID NO: 4-6). The internal reference substance and the detection primers can be used for preparation of a kit for detecting a bladder cancer serum internal reference substance and for detection on the bladder cancer serum miRNA internal reference.

Description

technical field [0001] The invention relates to an internal reference for bladder cancer serum miRNA detection, a detection primer and application thereof, and belongs to the technical field of molecular biology detection. Background technique [0002] Bladder cancer is a type of multiple urogenital tumors, and its morbidity and mortality in Chinese men are increasing year by year. About 30% of newly diagnosed bladder cancer patients are metastatic invasive bladder cancer (MIBC), while 30% of non-invasive bladder cancer (NMIBC) patients will recur as invasive bladder cancer. Early diagnosis and treatment can improve the therapeutic effect and prognosis of bladder cancer patients, so it is necessary to find practical diagnostic methods with high specificity and sensitivity. At present, the clinical methods for diagnosing bladder cancer mainly include cystoscopy, urinary exfoliation cytology and imaging methods. Among them, cystoscopy is a common method for diagnosing bladder...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/166C12Q2600/178
Inventor 王传新杜鲁涛刘益民张欣杨咏梅
Owner SHANDONG UNIV QILU HOSPITAL
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