A diagnostic marker for chronic transit constipation and its application
A chronic constipation, transport-type technology, applied in the direction of drug combination, biochemical equipment and methods, microbial determination/testing, etc.
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Embodiment 1
[0034] The collection of embodiment 1 sample and total RNA extraction
[0035] The left colon tissues of 3 patients with chronic transit constipation in the hospital were collected as the disease group, and the colon tissues of the left hemiparasinoma of 3 patients with colon cancer were collected as the control group (without symptoms of constipation). The patients did not receive radiotherapy and chemotherapy before the operation, and the materials were collected during the operation, stored in liquid nitrogen immediately, and then transferred to -80°C for long-term storage for RNA extraction.
[0036]RNA extraction standards: RNA purity: OD260 / 280≧1.8, 28S / 18S≧1; RNA integrity: RIN value≧7.0. RNA integrity detection method: Agilent 2100 (RNA 6000Nano kit), agarose gel electrophoresis (agarose gel concentration: 1% agarose gel; voltage: 5V / cm; time: 20min).
[0037] Table 1 Samples and their RNA extraction
[0038]
Embodiment 2
[0039] Example 2 Sequencing and Data Analysis
[0040] Sequencing: mRNA, miRNA, and lncRNA were sequenced using llumina Hiseq2500 / Miseq second-generation high-throughput sequencing technology, and data processing was completed by removing adapters, removing low-quality, and decontaminating processes to obtain final data. Analyze the sequencing data to quantify the expression levels of all known transcripts and compare whether there are differences in the expression of genes in different groups.
[0041] The sequencing platform is the HiSeq 2500 high-throughput sequencing platform of Illumina, which performs high-throughput transcriptome deep sequencing. After sequencing, we use Fast-QC software to evaluate the quality of the sequencing data as a whole, including the quality value distribution of bases, quality value position distribution, GC content, PCR duplication content, kmer frequency, etc.
[0042] In the analysis of differential mRNA and lncRNA expression, use software...
Embodiment 3
[0044] Example 3 Detection of the expression of the LOC102723345 gene in STC patients by Real-time PCR
[0045] 1 sample collection:
[0046] Peripheral blood was collected from 34 patients with chronic transit constipation and 29 healthy people as controls, and they were grouped and numbered.
[0047] 2 total RNA extraction:
[0048] Removal of RNase for related experimental items:
[0049] ① Rinse and soak all glassware with DEPC before use, 120°C for 20 minutes under high pressure, and 180°C for more than 2 hours.
[0050] ②Plastic utensils (such as: EP tubes / tips) need to be soaked in 0.1% DEPC water overnight before use, and then the liquid is controlled to dry, 120°C and high pressure for 20 minutes, and dried in an oven for later use.
[0051] leukapheresis
[0052] (1) Take 2m1 anticoagulated peripheral blood (blood collection time does not exceed 3h);
[0053] (2) Add an equal volume of sterile PBS to the peripheral blood and mix thoroughly to form a cell suspens...
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