Molecular marker for child respiratory syncytial virus infection and application of molecular marker
A technology of molecular markers and markers, applied in the field of biomedicine, can solve the problem of less research on the role of host factors
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Embodiment 1
[0045] Example 1 Screening for Gene Markers Related to Children's Respiratory Syncytial Virus Infection
[0046] 1. Sample collection
[0047] The blood samples of 5 normal children and children with respiratory syncytial virus infection were collected, and the sample name, number, sampling date, sample processing process, etc. were written. All the above samples were obtained with the consent of the ethics committee.
[0048] 2. RNA sample preparation and quality analysis
[0049] 2.1 Total RNA was extracted using the RNA extraction kit from Promega. Specific steps are as follows:
[0050] 1) Take 1ml of whole blood collected in a test tube treated with heparin or EDTA, put it into a sterile centrifuge tube, centrifuge at 3000rpm for 5min, and carefully suck off the supernatant from the top of the sample;
[0051] 2) Add 1ml of blood cell lysate, pipette carefully 4-5 times, resuspend the sediment, and centrifuge at 3000rpm for 5min;
[0052] 3) Repeat step 2) twice;
[...
Embodiment 2
[0073] Example 2 QPCR sequencing to verify the differential expression of genes
[0074] 1. According to the detection results of high-throughput sequencing, the differentially expressed genes were verified by large-sample QPCR. According to the sample collection method in Example 1, blood from 30 children with respiratory syncytial virus infection and 25 normal children were selected.
[0075] 2. RNA extraction steps are the same as in Example 1
[0076] 3. Reverse transcription:
[0077] (1) Reverse transcription reaction:
[0078] Use 25μl reaction system, take 1μg total RNA for each sample as template RNA, and add the following components to PCR tubes: DEPC water, 5× reverse transcription buffer, 10mM dNTP, 0.1mM DTT, 30μM Oligo dT, 200U / μl M-MLV, template RNA. Incubate at 42°C for 1 hour, then centrifuge briefly at 72°C for 10 minutes.
[0079] (2) Primer design
[0080] QPCR amplification primers were designed according to the coding sequences of H2AFJ and RGL4 gen...
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