Method for detecting long non-coding RNA in urine of prostate cancer patients by using digital PCR

A prostate cancer, digital application technology, applied in the field of tumor molecular biology, can solve the problems of difficulty in detecting prostate cancer, cumbersome operation and high cost, and achieve the effects of easy automatic detection, simple operation and low detection limit

Pending Publication Date: 2019-12-17
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Prostate cancer is often asymptomatic in the early stage, it is difficult to diagnose, and it is easy to delay the timing of treatment, which seriously endangers the health of men in my country
At present, the clinical diagnosis of prostate cancer mainly relies on digital rectal examination, serum PSA, transrectal ultrasound of the prostate and pelvic MRI examination. The above methods are difficult to detect early prostate cancer lesions, and digital rectal examination and transrectal ultrasound of the prostate bring pain to patients. Larger, blood sampling is required for serum PSA examination, and pelvic MRI examination has the disadvantages of cumbersome operation and high cost. Therefore, there is an urgent need for an early screening method for prostate cancer that is easy to operate, simple to sample, and harmless to patients

Method used

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  • Method for detecting long non-coding RNA in urine of prostate cancer patients by using digital PCR
  • Method for detecting long non-coding RNA in urine of prostate cancer patients by using digital PCR
  • Method for detecting long non-coding RNA in urine of prostate cancer patients by using digital PCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] The collection and pretreatment of embodiment 1 urine sample

[0066] The steps specifically include:

[0067] 1-1) The traditional prostate massage method is used, that is, the examiner performs a digital rectal examination, touches the prostate on the forearm of the rectum, massages the central groove three times symmetrically from the left and right sides, and then massages the central groove three times from the bottom of the prostate to the tip, and then instructs the patient Collect 50ml of initial urine after urination, and immediately cool it in cold water; within 2 hours after donation, transfer the urine (about 100ml) from the collection cup to a 50ml sterile centrifuge tube;

[0068] 1-2) Centrifuge at 4°C, 3000r / min for 5 minutes, discard the supernatant (be careful not to pour out the precipitate), and collect the urine sediment;

[0069] 1-3) Resuspend the precipitate with 1ml of pre-cooled PBS solution, transfer to a 1.5ml centrifuge tube, centrifuge at ...

Embodiment 2

[0071] Example 2 RNA extraction (extracted using invitrogenTRIzol reagent)

[0072] The steps specifically include:

[0073] 2-1) Add Trizol solution to the treated test sample prepared in Example 1, vortex and mix, and let stand for five minutes;

[0074] 2-2) Add 0.2ml chloroform to every 1ml Trizol solution, vortex and mix well and let stand for 2-3 minutes;

[0075] 2-3) Centrifuge at 12000g for 15 minutes at 4°C, and transfer the supernatant aqueous phase to a new 1.5ml centrifuge tube;

[0076] 2-4) Add 10 μg of RNase-free glycogen and 0.5 ml of isopropanol to the supernatant obtained per 1 ml of Trizol solution, vortex and mix well, and let stand for 10 minutes;

[0077] 2-5) Centrifuge at 4°C and 12000g for 10 minutes, discard the supernatant;

[0078] 2-6) Add 1ml of ethanol with a volume fraction of 75% to the precipitate obtained per 1ml of Trizol solution, and gently invert and mix;

[0079] 2-7) Centrifuge at 4°C and 7500g for 5 minutes, discard the supernatan...

Embodiment 3

[0082] Example 3 reverse transcription (using TAKARA reverse transcription kit)

[0083] The steps specifically include:

[0084] 3-1) Genomic DNA removal reaction: prepare the reaction mixture on ice according to the following components;

[0085] Reagent Usage amount 5*gDNA Eraser Buffer 2.0 μL gDNA Eraser 1.0 μL Total RNA (made by embodiment 2) 100ng RNase Free dH2O Make up to 10μL

[0086] Mix well and incubate at 42°C for 2 minutes to remove genomic DNA from total RNA; then place on ice;

[0087] 3-2) For the reverse transcription reaction, configure the reaction mixture on ice according to the following components;

[0088] Reagent Usage amount Step 3-1) reaction solution 10 μL PrimeScript RT Enzyme Mix I 1μL RT Primer Mix 1μL 5*PrimeScript Buffer 2 4μL RNase Free dH2O 4μL total 20 μL

[0089] After the preparation and mixing, carry out the reverse transcription react...

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Abstract

The invention discloses a method for detecting long non-coding RNA in urine of prostate cancer patients by using digital PCR. The method comprises the steps that the urine of an object to be detectedis used as a detection sample, and a digital PCR method is adopted to detect specific lncRNA of prostate cancer in urine. The method uses digital PCR to detect lncRNA in urine samples. The content oflncRNA in the urine samples is relatively low, and therefore compared with traditional fluorescence quantitative PCR, the method has the advantages of being more sensitive in detection, lower in detection limit and capable of performing absolute quantification. The method uses a urine sample of a patient for detection. Compared with a traditional blood sample or tumor tissue sample, the detectionsample is easier to obtain and less harmful to the patient. The method is simple in operation, procedures are simplified, repetition is easy, automatic detection is easy, the detection is sensitive, and the detection limit is low.

Description

technical field [0001] The invention relates to the field of tumor molecular biology, in particular to a method for detecting long non-coding RNA in urine of prostate cancer patients by using digital PCR. Background technique [0002] Prostate cancer refers to epithelial malignant tumors that occur in the prostate. In 2012, the incidence rate of prostate cancer in my country's tumor registration areas was 9.92 / 100,000, ranking sixth in the incidence rate of male malignant tumors. Prostate cancer is often asymptomatic in the early stage, it is difficult to diagnose, and it is easy to delay the timing of treatment, which seriously endangers the health of men in our country. At present, the clinical diagnosis of prostate cancer mainly relies on digital rectal examination, serum PSA, transrectal ultrasound of the prostate and pelvic MRI examination. The above methods are difficult to detect early prostate cancer lesions, and digital rectal examination and transrectal ultrasound...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/158C12Q2600/178C12Q2531/113C12Q2563/159
Inventor 尹焕才刘荻田晶晶袁通阔唐玉国
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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