Reagent kit for quantitatively assessing long-term recurrence risks of breast cancer
A long-term recurrence and quantitative evaluation technology, applied in the direction of material excitation analysis, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problems that affect the development of detection and less attention, and achieve stable detection results, low detection cost, and easy operation. simple and fast effect
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Embodiment 1
[0106] Embodiment 1, the selection of clinical specimen and RNA extraction
[0107] Paraffin samples from the primary site were taken, and qualified tissue samples were screened after HE staining of tissue sections, and samples with less than 5% tumor cells were excluded.
[0108] Total RNA was extracted from 6 paraffin tissue slices cut into 10 μm thick by Beijing Huamei Shengke Biotechnology Co., Ltd. (Qiagen, product number: 74106).
[0109] Total RNA needs to be treated with DNase I to remove DNA impurities.
[0110] use PCR is for β-actin DNA (ABI, product number: 401846) to detect whether there is any DNA residue, if there is any, it needs to be treated with DNase I again. Quantification of total RNA.
Embodiment 2
[0111] Embodiment 2, reverse transcription
[0112] The specific primer working solution concentration of 21 genes is 10 μM, 0.4 μl of reverse primer solution (final concentration is 200nM) is mixed for each of the 21 genes, 2 μl of 10×RT-PCR buffer (10 is the dilution factor), and dNTP is mixed Solution 4 μl (final concentration is 500nM), RNase inhibitor 1 μl (10 units), reverse transcriptase 1 μl (4 units), RNA template extracted in Example 1 50ng, and the remaining volume was supplemented with water.
[0113] Mix the upper amplification system evenly and incubate at 37°C for 60 minutes.
Embodiment 3
[0114] Embodiment 3, taqman-real-time fluorescent quantitative PCR
[0115] The 21 genes were detected by fluorescent quantitative PCR, and one PCR reaction was performed for each gene. The reaction system was prepared as follows: 200 nM each of the forward and reverse specific primers, 2 μl of 10×PCR buffer, 1.6 μl (200 nM) of dNTP mixture, 0.1 μl (0.5 units) of DNA polymerase, and 100 nM of taqman fluorescent probe , 0.8 μl of the reverse transcription product in Example 2 (equivalent to the reverse transcription product of 2ng RNA template), and the remaining volume was supplemented by water.
[0116] The amplification reaction was performed at 95° C. for 15 minutes, 95° C. for 10-15 seconds, 60° C. for 10-15 seconds, and 72° C. for 10-15 seconds, for 30 cycles, and 72° C. for 7 minutes. After the amplification reaction, the Ct value of each gene was recorded, representing the expression level of each gene.
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