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Application of CST1mRNA (Cystatin 1 Messenger RNA) and CST4mRNA (Cystatin 4 Messenger RNA)or proteins coded by CST1mRNA and CST4mRNA in preparation of bladder cancer markers and kit thereof

A kit and marker technology, applied in the field of diagnosis, can solve the problems of no bladder cancer correlation, etc., and achieve the effects of reducing sampling pain, good specificity, and convenient use

Active Publication Date: 2014-08-06
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no report on the correlation between CST1 and CST4 and bladder cancer

Method used

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  • Application of CST1mRNA (Cystatin 1 Messenger RNA) and CST4mRNA (Cystatin 4 Messenger RNA)or proteins coded by CST1mRNA and CST4mRNA in preparation of bladder cancer markers and kit thereof
  • Application of CST1mRNA (Cystatin 1 Messenger RNA) and CST4mRNA (Cystatin 4 Messenger RNA)or proteins coded by CST1mRNA and CST4mRNA in preparation of bladder cancer markers and kit thereof
  • Application of CST1mRNA (Cystatin 1 Messenger RNA) and CST4mRNA (Cystatin 4 Messenger RNA)or proteins coded by CST1mRNA and CST4mRNA in preparation of bladder cancer markers and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, magnetic bead method specifically enriches the mRNA of CST1, CST4 and SDHA gene from urine

[0039]According to the mRNA sequence design of CST1, CST4 and SDHA, capture the specific probe of CST1, CST4 and SDHA gene mRNA (the nucleotide sequence of CST1mRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of CST4mRNA is as shown in SEQ ID NO.2 , the nucleotide sequence of SDHA mRNA such as SEQ ID NO.3), specifically as follows: the probe for capturing CST1 mRNA is 5'-aaagagcacaactgtttcttctgca (dA) 30-3' (SEQ ID NO.4), and the probe for capturing CST4 mRNA is 5'-taccaggtctattagaagca(dA)30-3'(SEQ ID NO.5), the probe for capturing internal reference gene SDHA (ubiquinone reductase) mRNA is 5'-ggagcgaatggctggcgggacg(dA)30-3'(SEQ ID NO.5). 6), the above-mentioned specific probe can complementarily bind to the olig(dT) of magnetic beads (GE, product number: 3815-2103-010150) to obtain magnetic beads bound to the specific probe.

[0040] Enrich the mRNA o...

Embodiment 2

[0054] Embodiment 2, detecting the expression of CST1 and CST4 in bladder cancer tissue

[0055] 30 cases of bladder cancer and adjacent paired tissue samples were collected from the Urology Department of Shanghai Fifth People's Hospital, cut to the size of rice grains, stored in RNAlater preservation solution at -80°C, and equilibrated to room temperature before use. Then according to the method of Example 1, the CST1mRNA, CST4mRNA and SDHA mRNA of bladder cancer and paracancerous paired tissues were enriched respectively, and then 30 cases of bladder cancer and paracancerous samples were detected with CST1 detection primers, CST4 detection primers and internal reference gene SDHA detection primers respectively. The relative expression levels of CST1 gene and CST4 gene in paired tissues, the detection system and detection conditions are the same as those in Example 1. The test results are 2 -ΔΔCP The relative expression was calculated by the method, and then the ratio (C / N) ...

Embodiment 3

[0056] Embodiment 3, construct bladder cancer detection kit

[0057] 1. Construction of CST1 recombinant plasmid

[0058] The total mRNA of bladder cancer cell line T24 was extracted from bladder cancer cell line T24, and then cDNA was synthesized using the extracted mRNA as a template, and the primers for constructing CST1 recombinant plasmid were designed according to the CST1 gene sequence, and the upstream primer was 5'-ctggagccccaaggagga-3' (SEQ ID NO.13), the downstream primer is 5'-accagtccagggggtggga-3' (SEQ ID NO.14), with the nucleotides shown in SEQ ID NO.13 and SEQ ID NO.14 as primers, the synthetic cDNA is The template was amplified by PCR, and the amplification conditions were: denaturation at 94°C for 5 minutes; 45 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute; extension at 72°C for 5 minutes, and cooling at 4°C. The amplified product was connected to the pTZ57R vector to construct a CST1 rec...

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Abstract

The invention discloses an application of CST1mRNA (Cystatin 1 Messenger RNA) and CST4mRNA (Cystatin 4 Messenger RNA) or proteins coded by CST1mRNA and CST4mRNA in preparation of bladder cancer markers and a kit thereof. When the CST1mRNA and the CST4mRNA or the proteins coded by the CST1mRNA and the CST4mRNA are combined and used for diagnosing and predicting bladder cancer, the specificity and sensitivity are higher than those of one marker therein. The invention further discloses the kit for detecting the markers. The kit is convenient in use, can be used for directly collecting urine for detecting without collecting serum so as to reduce the sampling pain of patients, has the characteristics of good specificity and high sensitivity, and can be used for bladder cancer diagnosis, curative effect assessment in the treatment process and metastasis recurrence monitoring after treatment so as to provide a guidance for advanced intervention of doctors.

Description

technical field [0001] The invention belongs to the field of diagnosis, and in particular relates to the application of Cystatin SN and Cystatin S in preparing bladder cancer markers, and also relates to a kit for diagnosing bladder cancer. Background technique [0002] Bladder cancer is the most common malignant tumor of the urinary system and one of the ten most common tumors in the whole body. It occupies the first place in the incidence of genitourinary system tumors in my country, and ranks second in the West after prostate cancer. In 2012, the incidence rate of bladder cancer in the national tumor registration area was 6.61 / 100,000, ranking ninth in the incidence rate of malignant tumors. Bladder cancer can occur at any age, even in children. Its incidence rate increases with age, and the highest incidence age is 50-70 years old. With the acceleration of the aging process in our country, the incidence of bladder cancer is showing an increasing trend year by year. I...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/68G01N33/577
CPCC12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/158G01N33/57407G01N33/68G01N2333/8139
Inventor 王弢渠香云何林富
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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