Novel application of mir-3613 and mature miRNA thereof
A mature technology of mir-3613, applied in the field of genetic testing, can solve problems such as unsatisfactory prognosis, low survival rate, and poor prognosis
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Embodiment 1
[0052] The collection of embodiment 1 sample
[0053] The samples of 3 cases of metastatic esophageal squamous cell carcinoma tissue, 3 cases of non-metastasis esophageal squamous cell carcinoma tissue and 6 cases of paracancerous tissue were obtained from surgical resection specimens in the hospital from June 2015 to January 2017. The body was placed in a liquid nitrogen tank within 10 minutes, and then transferred to a -80°C refrigerator for storage.
Embodiment 2
[0054] Example 2 total RNA extraction
[0055] 1 extraction method
[0056] 1) Take 80 mg tissue block, add 800 μl Lysis / Binding buffer, and use a homogenizer to homogenize the tissue block. During the homogenization process, the samples should be placed on ice to keep the cold state.
[0057] 2) Add 1 / 10 volume of Homogenate Additive to the above homogenized tissue samples, and place on ice for 10 minutes.
[0058] 3) Add an equal volume of water-saturated phenol to the Lysis / Binding buffer, shake for 45 seconds, and centrifuge at 10,000×g for 5 minutes at room temperature.
[0059] 4) Carefully remove the supernatant into a new test tube, add 1.25 times the volume of absolute ethanol, mix well, transfer to a purification column, centrifuge at 10,000×g for 15 s, and discard the liquid in the collection tube. Since the maximum volume of the column is only 700 μl, repeat this step until all the supernatant is filtered.
[0060] 5) Add 700 μl of miRNA eluent 1 to the spin co...
Embodiment 3
[0069] Example 3 Sequencing and Data Analysis
[0070] The sequencing company carried out the establishment of the sequencing library and on-machine sequencing, and the sequencer used was the HiSeq2000 sequencer of Illumina Company.
[0071] Statistical analysis was performed according to the data provided by the sequencing company, fdr1, and the difference between the mean count values of the two groups was greater than 100. Differentially expressed miRNAs were artificially selected to filter miRNAs that were significantly differentially expressed, and several previously unreported molecular markers related to esophageal squamous cell carcinoma entered our research scope, among which miR-3613-5p was in the metastasis group and the diseased group. Although there was differential expression between the two groups, the difference was not significant, and the expression levels in metastatic and diseased cancer tissues were significantly higher than those in paracancerous tissue...
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