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

A technology for the detection of primers and bladder cancer, which is applied in DNA/RNA fragments, recombinant DNA technology, and the determination/examination of microorganisms. Stable and reliable performance, broad application prospects, and the effect of eliminating differences in expression levels

Active Publication Date: 2014-02-26
SHANDONG UNIV QILU HOSPITAL
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  • Summary
  • Abstract
  • 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 Specimens and Data Arrangement

[0044] The inventors of the present invention collected serum samples from 250 patients with bladder cancer from Qilu Hospital of Shandong University between 2011 and 2013, and collected serum samples from 158 controls 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, all of whom had not undergone surgery, radiotherapy and chemotherapy before blood collection.

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

[0047] The system collected the demographic data, clinicopathological data and other information of the above subjects.

Embodiment 2

[0048] Example 2 Solexa sequencing of serum miRNA

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

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

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

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

[0053] (4) Transfer the water phase to a new centrifuge tube, then add isopropanol at a volume ratio of 0.5ml isopropanol per 1ml Trizol reagent, let stand...

Embodiment 3

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

[0063] According to the results of Solexa sequencing, select miRNA molecules that meet the following criteria for further screening by qRT-PCR technology: a) the expression copy number is 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 among the three groups (p≥0.05). According to the above criteria, a total of 10 miRNA molecules meeting the requirements were selected (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, U6 was also included in the screening as a candidate molecule in order to verify whether it can be used as an internal reference in serum. The abo...

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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, detection primers and application thereof, and belongs to the technical field of molecular biology detection. Background technique [0002] Bladder cancer is a kind of multiple genitourinary system tumors, and its morbidity and mortality in Chinese men are increasing year by year. About 30% of newly diagnosed bladder cancer patients are invasive bladder cancer (MIBC) that is prone to metastasis, while 30% of non-invasive bladder cancer (NMIBC) patients will still relapse into invasive bladder cancer. Early diagnosis and treatment can improve the treatment effect and prognosis of bladder cancer patients, so it is necessary to find a practical diagnostic method with high specificity and sensitivity. At present, the clinical methods used to diagnose bladder cancer mainly include cystoscopy, urine exfoliation cytology, and imaging methods. Among them, cystoscopy is a ...

Claims

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Application Information

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Patent Type & Authority Applications(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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