Application of CST1mRNA and CST4mRNA or proteins encoded by CST4mRNA in preparing renal cancer markers and kit of markers

A kit and reagent technology, applied in the field of diagnosis, to achieve good specificity, convenient use, and reduce the pain of sampling

Active Publication Date: 2015-06-10
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 CystatinSN (CST1) and CystatinS (CST4) and renal cancer

Method used

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  • Application of CST1mRNA and CST4mRNA or proteins encoded by CST4mRNA in preparing renal cancer markers and kit of markers
  • Application of CST1mRNA and CST4mRNA or proteins encoded by CST4mRNA in preparing renal cancer markers and kit of markers
  • Application of CST1mRNA and CST4mRNA or proteins encoded by CST4mRNA in preparing renal cancer markers and kit of markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, magnetic bead method specifically enriches the mRNA of CST1, CST4 and RPN1 gene from urine

[0035] According to the mRNA sequence design of CST1, CST4 and RPN1 (ribosome binding protein 1, ribophorin1) capture the specific probe of the mRNA of CST1, CST4 and RPN1 gene (the nucleotide sequence of CST1mRNA is as shown in SEQ ID NO.1, the nucleotide sequence of CST4mRNA The nucleotide sequence such as SEQ ID NO.2, the nucleotide sequence of RPN1mRNA such as SEQ ID NO.3), specifically as follows: the probe for capturing CST1mRNA is 5'-aaagagcacaactgtttcttctgca(dA)30-3'(SEQ ID NO. 4), the probe for capturing CST4mRNA is 5'-taccaggtctattagaagca(dA)30-3'(SEQ ID NO.5), the probe for capturing internal reference gene RPN1mRNA is 5'-gatgagcttctcattctcaatgtacg(dA)30-3'(SEQ ID NO.5) NO.6). The above-mentioned specific probes can complementarily bind to olig(dT) of magnetic beads (GE, catalog number: 3815-2103-010150) to obtain magnetic beads bound to specific probes...

Embodiment 2

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

[0051] 30 cases of kidney 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 RPN1mRNA of kidney cancer and adjacent paired tissues were enriched respectively, and then the detection primers of CST1, CST4 and internal reference gene RPN1 were used to detect 30 samples of kidney cancer and adjacent pairs. The relative expression levels of CST1 gene and CST4 gene in tissues, the detection system and detection conditions are the same as those in Example 1. The test results are 2 -ΔΔCP The relative expression level was calculated by the method, and then the ratio of the relative expression level (C / N) between renal cancer and...

Embodiment 3

[0052] Embodiment 3, construction kidney cancer detection kit

[0053] 1. Construction of CST1 recombinant plasmid

[0054] The total mRNA of kidney cancer cell line T24 was extracted from the kidney 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: pre-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 ...

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Abstract

The invention discloses an application of CST1mRNA and CST4mRNA or proteins encoded by CST4mRNA in preparing renal cancer markers and a kit of the markers. The CST1mRNA is used in combination with CST4mRNA or proteins encoded by CST4mRNA for the diagnosis and indication of renal cancer, so that the specificity and sensitivity higher than the case that one of markers is used; the invention also discloses a kit for detecting the marker, the kit is convenient to use, the detection can be carried out by directly collecting urine instead of serum and thus the sampling pain of the patient is reduced; the kit has the characteristics of good specificity and high sensitivity and can be used in diagnose of renal cancer, therapeutic effect evaluation in treatment process and monitoring of recurrence and metastasis after treatment, thereby providing a guidance for advanced interfere of doctors.

Description

technical field [0001] The invention belongs to the field of diagnosis, and specifically relates to the application of CST1mRNA and CST4mRNA or their coded proteins Cystatin SN and Cystatin S in the preparation of renal cancer markers, and also relates to a kit for diagnosing renal cancer. Background technique [0002] Renal cell carcinoma (referred to as renal cell carcinoma) accounts for about 2%-3% of malignant tumors in adults, and is the second most common malignant tumor of the urinary system in my country, second only to bladder cancer. Surgical resection is the most effective treatment for localized RCC, but many patients eventually relapse. The vast majority of renal cell carcinomas have no clinical symptoms in the early stage, and some patients have locally advanced or distant metastases when they are first diagnosed, and cannot be radically removed by surgery, and the prognosis of these patients is poor. Therefore, early diagnosis of kidney cancer becomes particu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N33/68G01N33/577
CPCC12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/158G01N33/57438G01N33/68G01N2333/8139
Inventor 王弢渠香云何林富高鹏
Owner SHANGHAI LIANGRUN BIOMEDICINE TECH CO LTD
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