Gene chip for detecting functional central nerve damage and its production method
A central nervous system and gene chip technology, applied in the field of genes, can solve the problems of high cost and large amount of data, and achieve the effect of simple research work, speed up and progress
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Embodiment 1
[0040] Example 1: Preparation of Gene Chip for Diagnosis and Detection of Functional Central Nervous Injury
[0041] The construction of a.DNA square matrix: 381 kinds of specific genes and negative controls (bacterial control probes or human housekeeping genes (Housekeeping Genes)) closely related to central nervous system damage listed in Table 1 are randomly classified according to their functions Arrangement (in the computer detection program of the chip, there is a list of 381 gene probes generated according to the random arrangement sequence of Oligo probes on the spotting machine) on the DNA-ReadyType II Slide glass slide produced by Clontech Company in the United States. The slide has been processed by combinatorial chemistry, which not only provides efficient nucleic acid binding capacity, but also prevents cross-contamination between spots to the greatest extent.
[0042] b. Probe design and in situ synthesis: According to two important parameters of the gene chip: d...
Embodiment 2
[0044] Example 2: Application of gene chip for diagnosis and detection of functional central nervous system injury
[0045] a. RNA extraction: Use Trizol reagent, chloroform, isopropanol, and 75% alcohol to extract RNA from serum, cerebrospinal fluid, or damaged tissue. After repeated centrifugation, filtration, and washing, the obtained RNA is stored at 4°C until use.
[0046] b. Reverse transcription into cDNA: After mixing RNA with amine-modified random primers (such as 2-hydrogen adenine), RNase inhibitors, aa-dUTP / dNTPs, DTT and SSII Reverse Transcriptase for about 2 hours, use EDTA (pH8 .0) Stop the reaction and then hydrolyze the RNA with NaOH. The reverse-transcribed cDNA was extracted by MinElute filter column produced in the United States, and the purified cDNA was obtained after repeated centrifugation, filtration, washing and drying.
[0047] c. Fluorescent labeling: the sample cDNA was labeled with a fluorescent marker (Cy5 or Cy3) using a Qia-quick filter column...
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