本报告描述了一种快速可靠的方法来验证细胞 miRNAs 的 mRNA 目标。该方法采用合成生物素化锁定核酸 (低噪声放大) miRNA 模拟捕获靶 mRNA。随后, 采用链亲和素包覆磁性微珠对靶 mRNA 进行 qPCR 聚合酶链反应的定量。
Access restricted. Please log in or start a trial to view this content.
Access restricted. Please log in or start a trial to view this content.
Access restricted. Please log in or start a trial to view this content.
Access restricted. Please log in or start a trial to view this content.
Access restricted. Please log in or start a trial to view this content.
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 细胞系 - HEK293T 细胞 | ATCC | CRL-3216 | |
| 热灭活胎牛血清 (Hi-FBS) | GIBCO/Thermofisher | 10438-026 | |
| Dulbecco's 修改后的鹰's 培养基 (DMEM) | GIBCO/Thermofisher | 11995-065 | |
| 磷酸盐缓冲盐水 (PBS) (1x) | GIBCO/Thermofisher | 20012-027 | |
| 胰蛋白酶-EDTA (0.25%) | GIBCO/Thermofisher | 25200-056 | |
| 青霉素-链霉素溶液 (100x) | Cellgro/Mediatech | 30-002-CI | |
| DNase | Ambion | AM2238 | |
| Opti-MEM 减血清培养基 | GIBCO/Thermofisher | 3198-088 | |
| Liopfectamine 2000 转染试剂 | Invitrogen/Thermofisher | 11668-019 | |
| 生物素化 hsa-miR-125b | Exiqon | 339178/ID-27281622 | |
| 生物素化加扰对照 | Exiqon | 479997-671/ID-714884 | |
| IGEPAL | Sigma-Aldrich | 18896 | |
| 链霉亲和素磁珠 | Pierce | 88816 | |
| 酵母 tRNA | Invitrogen/Thermofisher | 15401-011 | |
| 牛血清白蛋白 (BSA) | Sigma-Aldrich | A2153 | |
| 氯化钾 (KCl) 溶液 | Sigma-Aldrich | 60142 | |
| 氯化镁 (MgCl2)溶液 | Sigma-Aldrich | M1028 | |
| 氢氧化钠 (NaOH) | Sigma-Aldrich | 795429 | |
| 乙二胺四乙酸二钠盐 (EDTA) 溶液,0.5 M,pH 8.0 | Sigma-Aldrich | E7889 | |
| 二硫苏糖醇 (DTT) | Sigma-Aldrich | 43815 | |
| 超级酶 | Ambion/Thermofisher | AM2694 | |
| 蛋白酶抑制剂混合物 | Sigma-Aldrich | P8340 | |
| 不含 RNAse/DNAse 的水 | GIBCO/Thermofisher | 10977-015 | |
| RNeasy 提取试剂盒 | Qiagen | 74104 | |
| iScript-Select cDNA 合成试剂盒 | Biorad | 170-8897 | |
| qPCR 引物 | Invitrogen/Thermofisher | ||
| iTaq-Universal SYBR 绿色超混合液 | Biorad | 172-5120 | |
| DynaMag-Spin 磁力架 | Invitrogen/Thermofisher | 12320D |
Access restricted. Please log in or start a trial to view this content.
Request permission to reuse the text or figures of this JoVE article
Request Permission