实验步骤
1、首先将上海净信的组织研磨机上的制冷模块预先置于适当的温度下,使用时取出安装好。将研磨珠去RNA酶处理。
2、随机抽取小鼠的尾巴,把样本剪成尽可能小段,置于无RNA酶的离心管中(选择耐液氮冷冻管子),同时加入几颗研磨珠。
3、放置于液氮中一起浸泡几分钟,取出放置于预冷模块中,在全自动样品研磨仪上选择合适的研磨时间和研磨频率。(该步骤可以根据研磨的细微程度要求可以再重复一遍)
4、把研磨好的样本加入的PBS和RNAiso Plus(此处选择PBS,可以根据实验需要加入其他提取液,如TRIzol等),继续置于研磨机上选择合适的研磨时间和研磨频率。样本将处于糊状。(其他提取试剂有可能会出现泡沫,不影响实验)
5、取出研磨管,放入冷冻离心机中,选择适当的温度进行离心几分钟。
6、小心吸取上清液至新的离心管中,盖上管盖,在室温上孵育十几分种,使样品充分裂解至匀浆液较透明,如果仍有少量颗粒属于正常情况,不影响RNA提取质量和得率。
7、在离心管中加入氯仿,盖紧管盖,振荡混匀并将其在冰上孵育几分种,选择适当的温度进行离心几分钟。离心后混合物分层:上清层,中间层,下层;RNA存在于上清层中。
8、将上清层小心转移到一干净的离心管中,加入等体积冰浴的异丙醇,颠倒振荡混匀,将混合的样品在适当的温度条件下,孵育几分种,选择适当的温度进行离心几分钟。RNA沉淀通常形成片状沉淀附着于管壁或管底。
小心弃去上清,用冰浴的乙醇洗涤RNA沉淀一次,颠倒洗涤离心管管壁,并旋涡振荡样品,尽可能让沉悬浮,选择适当的温度进行离心几分钟,再次去除上清,晾干沉淀。
10、操作的最后,在阴凉处适度干燥RNA沉淀(避免过分干燥,那样会降底它的可溶性)。
11、用适量的无RNA酶水或TE溶液来溶解RNA。抽提好的RNA,保存于适当的温度下。
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