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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 urethral carcinoma markers and kit thereof

A kit and technology for urethral cancer, applied in the field of diagnosis, can solve problems such as no correlation with urethral cancer, and achieve the effects of reducing sampling pain, good specificity, and convenient use

Active Publication Date: 2014-08-06
SHANGHAI LIANGRUN 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 urethral 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 urethral carcinoma 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 urethral carcinoma 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 urethral carcinoma markers and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, magnetic bead method specifically enriches the mRNA of CST1, CST4 and SDHA gene from urine

[0035] According to the mRNA sequence design of CST1, CST4 and SDHA, the specific probe (the nucleotide sequence of CST1 mRNA is shown in SEQ ID NO.1, the nucleotide sequence of CST4 mRNA is 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), the probe for capturing CST4 mRNA It is 5'-taccaggtctattagaagca (dA) 30-3' (SEQ ID NO.5), and the probe for capturing internal reference gene SDHA (ubiquinone reductase) mRNA is 5'-ggagcgaatggctggcgggacg (dA) 30-3' (SEQ ID NO. .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.

[0036]Enrich the mRNA of CST1, CST4 and SDHA genes, the specific st...

Embodiment 2

[0050] Example 2, detection of CST1 and CST4 expression in urethral cancer tissue

[0051] 30 paired tissue samples of urethral cancer and adjacent tumors 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 the paired tissues of urethral cancer and adjacent cancer were respectively enriched, and then 30 cases of samples of urethral cancer and adjacent cancer 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 using the method, and then t...

Embodiment 3

[0052] Embodiment 3, construction urethral cancer detection kit

[0053] 1. Construction of CST1 recombinant plasmid

[0054] The total mRNA of the urethral cancer cell line T24 was extracted from the urethral cancer cell line A498, and cDNA was synthesized using the extracted mRNA as a template, and the primers for constructing the 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 constr...

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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 urethral carcinoma markers and a kit thereof. When the CST1mRNA and the CST4mRNA or the proteins coded by the CST1mRNA and the CST4mRNA are used for diagnosing and predicting urethral carcinoma, 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 urethral carcinoma 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 specifically relates to the application of Cystatin SN and Cystatin S in preparing urethral cancer markers, and also relates to a kit for diagnosing urethral cancer. Background technique [0002] Urethral cancer is a urothelial tumor that can occur in both sexes. It is relatively rare clinically, and most of them are males. It is the most common tumor in the urethral tumor and is a malignant tumor. According to its origin, it is divided into squamous epithelial carcinoma, transitional epithelial carcinoma, adenocarcinoma, clear cell carcinoma, urethral cloacal carcinoma, adenoid cystic carcinoma, carcinoid, mixed carcinoma, cowper urethral accessory adenocarcinoma. Squamous cell carcinoma is the most common, mostly occurring in the anterior or middle urethra, transitional epithelial carcinoma mostly occurs in the posterior urethra, and adenocarcinoma mostly occurs in the middle urethra. Urethral ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/68G01N33/574
CPCC12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/158G01N33/57407G01N33/68G01N2333/8139
Inventor 王弢渠香云何林富高鹏
Owner SHANGHAI LIANGRUN BIOMEDICINE TECH CO LTD
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