實(shí)驗(yàn)步驟
1、首先將上海凈信的組織研磨機(jī)上的制冷模塊預(yù)先置于適當(dāng)?shù)臏囟认?,使用時(shí)取出安裝好。將研磨珠去RNA酶處理。
2、隨機(jī)抽取小鼠的尾巴,把樣本剪成盡可能小段,置于無(wú)RNA酶的離心管中(選擇耐液氮冷凍管子),同時(shí)加入幾顆研磨珠。
3、放置于液氮中一起浸泡幾分鐘,取出放置于預(yù)冷模塊中,在全自動(dòng)樣品研磨儀上選擇合適的研磨時(shí)間和研磨頻率。(該步驟可以根據(jù)研磨的細(xì)微程度要求可以再重復(fù)一遍)
4、把研磨好的樣本加入的PBS和RNAiso Plus(此處選擇PBS,可以根據(jù)實(shí)驗(yàn)需要加入其他提取液,如TRIzol等),繼續(xù)置于研磨機(jī)上選擇合適的研磨時(shí)間和研磨頻率。樣本將處于糊狀。(其他提取試劑有可能會(huì)出現(xiàn)泡沫,不影響實(shí)驗(yàn))
5、取出研磨管,放入冷凍離心機(jī)中,選擇適當(dāng)?shù)臏囟冗M(jìn)行離心幾分鐘。
6、小心吸取上清液至新的離心管中,蓋上管蓋,在室溫上孵育十幾分種,使樣品充分裂解至勻漿液較透明,如果仍有少量顆粒屬于正常情況,不影響RNA提取質(zhì)量和得率。
7、在離心管中加入氯仿,蓋緊管蓋,振蕩混勻并將其在冰上孵育幾分種,選擇適當(dāng)?shù)臏囟冗M(jìn)行離心幾分鐘。離心后混合物分層:上清層,中間層,下層;RNA存在于上清層中。
8、將上清層小心轉(zhuǎn)移到一干凈的離心管中,加入等體積冰浴的異丙醇,顛倒振蕩混勻,將混合的樣品在適當(dāng)?shù)臏囟葪l件下,孵育幾分種,選擇適當(dāng)?shù)臏囟冗M(jìn)行離心幾分鐘。RNA沉淀通常形成片狀沉淀附著于管壁或管底。
小心棄去上清,用冰浴的乙醇洗滌RNA沉淀一次,顛倒洗滌離心管管壁,并旋渦振蕩樣品,盡可能讓沉懸浮,選擇適當(dāng)?shù)臏囟冗M(jìn)行離心幾分鐘,再次去除上清,晾干沉淀。
10、操作的最后,在陰涼處適度干燥RNA沉淀(避免過(guò)分干燥,那樣會(huì)降底它的可溶性)。
11、用適量的無(wú)RNA酶水或TE溶液來(lái)溶解RNA。抽提好的RNA,保存于適當(dāng)?shù)臏囟认隆?/p>
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