Intestinal tissue marker lncRNAH19 after severe burn and its application
A technology for severe burns and intestines, which is applied in the field of intestinal tissue marker lncRNAH19 after severe burns, and can solve problems such as the difficulty in completely repairing the intestinal mucosal barrier
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Embodiment 1
[0010] Example 1 Screening for markers related to intestinal tissue repair after severe burns
[0011] 1. Sample collection
[0012] Clean grade adult 20-25g C57BL / 6J mice were selected as research objects. Burn model production: pentobarbital sodium 1% pentobarbital sodium (0.1ml / 10g) was injected intraperitoneally, the back was depilated with 8% sodium sulfate, and the back was placed in 100°C boiling water for 10 seconds, resulting in 30% TBSAⅢ° scald, and the abdominal cavity was immediately burned. Inject sodium lactate Ringer's injection (50ml / kg) for anti-shock treatment, rub povidone iodine on the back for anti-infection, and cover with gauze to keep warm. Sham injury model: place the back in a water bath at 37°C for 10 seconds.
[0013] 1. RNA extraction
[0014] 1) Take 50-100mg of small intestinal ileocecal tissue, put it on ice and add 1ml Trizol, use a homogenizer to fully homogenize the tissue, transfer it to a new 1.5ml nuclease-free EP tube, and let it stand...
Embodiment 2
[0029] Example 2 qRT-PCR sequencing to verify the differential expression of related genes
[0030] 1. Use the chip to analyze the remaining RNA samples (N=3 in the burn group, N=3 in the control group) to perform QPCR verification on six lncRNAs.
[0031] 2. Reverse transcription:
[0032] 1) Configuration of reverse transcription system:
[0033] Use 20ul reaction system, take 2ug total RNA for each sample as RNA template, 2ulRandom, 2ulSuperPure dNTPs (2.5mM each), supplement RNase-Free ddH 2 O was fixed to 14.5ul. After heating at 70°C for 5 minutes, it was rapidly cooled on ice for 2 minutes.
[0034] After brief centrifugation to collect the reaction solution, the following components were added: 4ul 5x First-Strand Buffer (containing DTT), 0.5ul RNasin, 1ul (200U) TIANScript M-MLV.
[0035] 2) Reverse transcription reaction conditions:
[0036] 10min at 25°C, 50min at 42°C, 5min at 95°C, and store on ice for subsequent experiments or cryopreservation.
[0037] 3) QP...
Embodiment 3
[0049] Example 3 qRT-PCR sequencing to verify the differential expression of the lncRNA-H19 gene:
[0050] 1. Large-sample QPCR verification of H19 gene differential expression. The ileocecal intestinal tissue after severe burn (N=20) and the ileocecal intestinal tissue (N=15) of the control group were selected according to the sample collection method in (1).
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