Kit for screening steroid-induced necrosis of femoral head
A femoral head necrosis and kit technology, applied in the fields of biomolecules and medicine, can solve the problems that cannot fully explain the pathological process of femoral head necrosis
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Embodiment 1
[0031] Example 1 High-throughput sequencing screening of differentially expressed genes
[0032] 1. Sample
[0033] Collect blood samples from 5 patients with steroid-induced necrosis of the femoral head, numbered F1-F5; collect blood samples from 3 relatively healthy subjects. Number N1 suffered from traumatic necrosis of the femoral head, and N2 and N3 suffered from acetabular dysplasia. The samples were collected from patients who visited the Department of Orthopedics of Peking Union Medical College Hospital from October 2015 to May 2018. All clinical samples in this study were informed to the patients and approved by the Hospital Ethics Committee.
[0034] 2. Extract total RNA from the sample
[0035] 2.1 RNA extraction sample processing
[0036] Mix EDTA anticoagulated whole blood with Trizol at a ratio of 1:1. If RNA extraction is not to be performed within a short period of time, the sample can be quickly frozen in liquid nitrogen for 30s-1min, and stored at -80°C f...
Embodiment 2
[0051] Example 2 Real-Time PCR verification of miR-100-5p expression in patients with steroid-induced necrosis of the femoral head
[0052] 1. Materials
[0053] Fifteen blood samples from patients with steroid-induced necrosis of the femoral head and 5 blood samples from normal subjects were selected, and grouped and numbered. All samples were confirmed by pathological examination.
[0054] 2. See Example 1 for RNA extraction steps
[0055] 3. Reverse transcription
[0056] Using total RNA as a template, miRNA cDNA was synthesized using Transcript Reverse Transcriptase and Ribonuclease Inhibitor reverse transcription kits.
[0057] Synthesize the first cDNA strand:
[0058] Using a microvolume EP tube, add the following system A (12 μL) in sequence:
[0059] Table 1 System A
[0060] RNA (2μg)
XμL(x≤9)
miRNART Primer (RPrimer)
1μL
sonRNA202R Primer
1μL
dNTP (2.5mM)
1μL
DEPCwater
9-x μL
[0061] Mix gently, p...
Embodiment 3
[0079] Embodiment 3 kit preparation
[0080] Based on the primers obtained in Example 2, assemble the test kit for the prognostic diagnosis of steroid-induced necrosis of the femoral head according to the present invention, the kit includes (1) containing reverse transcriptase buffer, reverse transcription primers, reverse transcriptase, etc. Reverse transcription reagent; (2) The forward primer for specific amplification of miR-100 is shown in SEQ ID NO: 1, and the reverse primer is a universal primer such as SEQ ID NO: 2: and specific amplification of snRNA T6 (as an internal reference) The primer pair is shown as SEQ ID NO:5 and SEQ ID NO:6; (3) also includes SYBR Green polymerase chain reaction system, such as PCR buffer, SYBR Green fluorescent dye, dNTPs. The composition of described PCR buffer is 25mM KCl, 2.5mM MgCl 2 , 200mM (NH4) 2 SO 4 .
[0081] Through the optimization of primer concentration and annealing temperature, the reaction system was finally determined...
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