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Method for detecting microRNA expression level in excrement and selection method of internal reference gene

An internal reference gene and expression level technology, applied in the field of bioengineering, can solve problems such as microRNA detection that have not been reported, and achieve stable and reliable results, high repeatability, and overcoming unstable operation.

Inactive Publication Date: 2011-05-25
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

[0006] Based on the above studies, most of the studies on microRNA are limited to blood diseases, such as lung cancer, hepatocellular carcinoma, breast cancer, and colorectal cancer. There are very few studies on pancreatic cancer, and the research on pancreatic cancer is also limited to tissue, blood and other samples. , has not reported the detection of microRNA in feces

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  • Method for detecting microRNA expression level in excrement and selection method of internal reference gene
  • Method for detecting microRNA expression level in excrement and selection method of internal reference gene
  • Method for detecting microRNA expression level in excrement and selection method of internal reference gene

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Embodiment 1

[0031] Now take the detection of fecal microRNAs from patients with pancreatic cancer as an example to describe the invention in detail. This example is only for illustrating the present invention and is not intended to limit the scope of the present invention.

[0032] Selected 29 cases of pancreatic cancer and 22 cases of chronic pancreatitis admitted to Changhai Hospital from 2008 to 2010, and collected 13 cases of healthy controls in our laboratory.

[0033] 1. After the specimens are collected in cryopreservation tubes, place them in an ice box immediately, and transfer them to an ultra-low temperature freezer at -80°C within 6 hours for storage. During the RNA extraction of feces, 200 mg of feces should be weighed from the cryopreservation tube, and the total RNA (including microRNA) should be extracted by E.Z.N.A feces extraction kit (OMEGA, USA) to extract total microRNAs. The specific steps

[0034] as follows:

[0035] Weigh 200mg of stool sample into a 2ml centrif...

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Abstract

The invention discloses a method for detecting the microRNA expression level in excrement. The method mainly comprises the following steps: extracting with an excrement extraction kit from a collected excrement specimen, identifying the concentration of RNA with a NanoDrop; performing reverse transcription to generate cDNA, detecting the expression of the target microRNAs through Real-time RT-PCR; and adopting Real Time PCR to obtain the CT value of each microRNA, adopting the -Delta Delta Ct method to calculate the expression abundance of each microRNA, and selecting an internal reference gene. The invention also provides a selection method of the internal reference gene used for detecting pancreatic cancer. The detection method provided by the invention has reliable result and good stability.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for detecting the expression level of microRNA in feces and a method for selecting internal reference genes. Background technique [0002] microRNA (miRNA) molecules are research hotspots in the fields of molecular biology and genetics in recent years. miRNAs are a newly discovered class of small non-coding RNA molecules that usually regulate gene expression at the post-transcriptional level. The miRNA gene is first transcribed to produce a primary product (pri-miRNA) of hundreds to thousands of nucleotides, which is cut into a hairpin precursor of about 70 nucleotides ( pre-miRNA), pre-miRNA is transported out of the nucleus under the action of the transport protein Exortin=5, and then cut into about 22nt mature miRNA by Dicer enzyme (Gregory RI, Shlekhattar R.MicroRNA biogenesis and cancer.Cancer Res, 2005, 65( 9): 3509-3512.). Mature miRNAs form the RNA-indu...

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李兆申任艳高军王小玮刘建强顾骏俊
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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