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Polymerase chain reaction detection method of urinary podocyte specific messenger ribonucleic acid (mRNA)

A technology of messenger RNA and detection method, which is applied in the field of urine podocyte specific messenger RNA polymerase chain reaction detection, and achieves the effects of high specificity, high sensitivity and characteristic type, and good stability

Inactive Publication Date: 2012-05-09
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Urine contains a large amount of biological information and has the advantages of non-invasive collection and relatively simple specimen processing. However, there is no report on the construction of a method for detecting urine podocyte-specific mRNA based on real-time PCR technology.

Method used

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  • Polymerase chain reaction detection method of urinary podocyte specific messenger ribonucleic acid (mRNA)
  • Polymerase chain reaction detection method of urinary podocyte specific messenger ribonucleic acid (mRNA)
  • Polymerase chain reaction detection method of urinary podocyte specific messenger ribonucleic acid (mRNA)

Examples

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Effect test

Embodiment 1

[0018]Example 1: Construction and detection of urine podocalyxin, CD2-AP mRNA detection method: Step 1, collection of urine specimens: take 200-300ml of morning urine, put the morning urine on a centrifuge and keep the centrifugal force at 3000g and temperature at 4°C Centrifuge for 30 minutes, discard the supernatant, and resuspend the cell sediment in 1ml diethyl pyrocarbonate-PBS buffer (diethyl pyrocarbonate and PBS buffer are prepared at a volume ratio of 1:1000) to obtain a suspension. The liquid was placed on a centrifuge and centrifuged for 5 minutes at a centrifugal force of 12000g and a temperature of 4°C, discarded the supernatant, added 1ml RNAiso Plus (TAKARA, Dalian, China) to the remaining sediment, and mixed well to obtain a mixture of the sediment and RNAiso Plus. solution; step 2, total RNA extraction step: step 2.1 put the mixture of the sediment and RNAiso Plus on a centrifuge and centrifuge for 5 minutes at a centrifugal force of 12000g and a temperature of...

Embodiment 2

[0024] Example 2: Comparison of urinary podocalyxin and CD2-AP mRNA expression differences between diabetic nephropathy patients and healthy controls: 39 patients with clinical type 2 DN were selected, and DN was diagnosed based on history of type 2 diabetes, albuminuria, and diabetic retinopathy. Exclusion criteria: infection, tumor, trauma, surgery, hyperthyroidism, and other known kidney diseases. At the same time, 12 healthy adults without hypertension and diabetes were selected as controls. Step 1. Collection of urine specimens: take 200-300ml of morning urine, put the morning urine on a centrifuge and centrifuge for 30 minutes at a centrifugal force of 3000g and a temperature of 4°C, discard the supernatant, and resuspend the cell sediment in 1ml coke Diethyl carbonate-PBS buffer (diethyl pyrocarbonate and PBS buffer are prepared in a volume ratio of 1:1000) to obtain a suspension, which is placed on a centrifuge and centrifuged at a centrifugal force of 12000g and a tem...

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Abstract

The invention provides a polymerase chain reaction detection method of urinary podocyte specific messenger ribonucleic acid (mRNA). The method is characterized by being established through the following steps of: collection of urine specimens; total RNA (ribonucleic acid) extraction as well as concentration and purity detection; reverse transcription; design and synthesis of primers; real-time PCR (polymerase chain reaction); preparation of a standard product and manufacture of a standard curve; and real-time PCR of the specimens to be detected and the standard product. The method has the characteristics of rapidness, simplicity and convenience, and can be used for acquiring an excellent quantitative linear correlation coefficient (R2>0.99); simultaneously, the established method is wide in detectable concentration range, podocalyxin and CD2-PA respectively spans 10 orders of magnitude and 8 orders of magnitude, and Ct values corresponding to minimum detectable concentrations of two types of genes respectively reach 35 and 33.2286; and furthermore, the inter-group and inter-batch differences of the high-concentration and light-concentration template Ct values, detected by the method, of the two types of genes are less than 3%, thus indicating that the method is high in stability and good in repeatability. The established method can be used for providing a stable technical platform for rapidly, simply and conveniently detecting the urinary podocyte specific mRNA.

Description

technical field [0001] The invention belongs to the establishment of a research method for urinary biomarkers of diabetic nephropathy, in particular to the construction of a urine podocyte-specific messenger ribonucleic acid (mRNA) detection technology based on the SYBR Green I real-time fluorescent quantitative PCR method. Background technique [0002] Diabetic nephropathy (Diabetic nephropathy, DN) is the leading cause of end-stage renal disease (ESRD) in western developed countries. In my country, DN is the second cause of ESRD after glomerular diseases. The pathogenesis of DN has not yet been fully elucidated. Podocytes are a class of highly differentiated epithelial cells that attach to the glomerular basement membrane and are involved in maintaining the integrity of the filtration barrier. A large number of recent studies have confirmed that podocyte injury plays an extremely important role in the pathogenesis and progression of DN. Foot process fusion, podocyte hyp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 刘必成郑敏
Owner SOUTHEAST UNIV
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