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A brief version of this protocol appeared in:
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果蝇总RNA制备   

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摘要:主要介绍制备果蝇总RNA的方法,为后面的RNA实验做准备。

关键词: 果蝇, 总RNA, 制备

材料与试剂

  1. 1.5 ml离心管
  2. 果蝇
  3. TRIzolTM Reagent (InvitrogenTM, catalog number: 15596026)
  4. TURBO DNA-freeTM Kit (InvitrogenTM, catalog number: AM1907)
  5. 异丙醇
  6. 乙醇
  7. DEPC-H2O

仪器设备

  1. 离心机 (Eppendorf)
  2. Thermomixer compact (Eppendorf)

实验步骤

注:该实验需要在RNA洁净区操作,RNA易降解,需要使用RNA专用试剂耗材和设备。务必佩戴洁净的手套甚至口罩。

  1. 取200 μl TRIzol置于1.5 ml离心管中,放在冰上,将麻醉的10只果蝇放入冰上的离心管中,将果蝇碾碎,再加入800 μl TRIzol试剂,颠倒混匀。
  2. 室温放置10 min,间歇混匀数次。
  3. 4 °C,12,000 × g离心10 min,去掉果蝇残体,取上清至新管。
  4. 加入200 μl氯仿,混匀,室温放置5 min。
  5. 4 °C,12,000 × g离心15 min。
  6. 取上清 (约500 μl) 到新的RNAase free的离心管中,加入等体积的异丙醇混匀,室温放置10 min。
  7. 4 °C,12,000 × g离心30 min。
  8. 弃上清,加入500 μl 70%乙醇,颠倒数次,使RNA漂浮。
  9. 4 °C,7,500 × g离心5 min。
  10. 弃乙醇,如果残留的乙醇有挂壁现象,倒掉液体后再短暂离心,将残液用移液器吸出。室温放置直到白色沉淀呈透明装时加入50 μl DEPC-H2O溶解。
  11. 50 °C,600 rpm金属浴震荡混匀10 min或者时不时轻弹混匀。
  12. 加入1 μl DNase,5 μl DNase Buffer,37 °C温育30 min。
  13. 加5 μl DNase inactive reagent,室温放置5 min,间歇混匀2~3次。
  14. 4 °C,10,000 × g离心1.5 min。
  15. 转移上清至新的EP管中,测浓度;-80 °C保存。

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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:邓娟, 刘南. (2019). 果蝇总RNA制备. Bio-101: e1010254. DOI: 10.21769/BioProtoc.1010254.
How to cite: Deng, J and Liu, N. (2019). Preparation of Drosophila Total RNA. Bio-101: e1010254. DOI: 10.21769/BioProtoc.1010254.
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Q&A
By submitting a question/comment you agree to abide by our Terms of Service. If you find something abusive or that does not comply with our terms please contact us at eb@bio-protocol.org.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

鼎 裘
IBP
有没有果蝇total RNA 跑琼脂糖胶的质量鉴定图
3/9/2021 4:19:32 PM Reply
Nan Liu
Chinese Academy of Sciences, China

通过这个portal 我没有办法upload 胶图。

注意:果蝇total RNA的带型和哺乳动物不一样。其中果蝇的ribosome RNA会被切割。

刘南

3/11/2021 6:42:19 PM Reply


Bio-protocol editorial assistant
Bio-protocol journal

刘老师,

感谢您为读者解答!

您可以把胶图以图片形式直接在这回复框上传,或发给到这个邮箱eb@bio-protocol.org)我们来替您上传。

Bio-protocol 编辑部

3/11/2021 9:50:05 PM Reply


鼎 裘
IBP

没想到会收到您的回复,非常感谢您。这个问题是基于我们在提取果蝇total RNA时,大多数情况下跑胶鉴定只能看见2条rRNA亮带,28S的条带较弱,在这种情况下,我们进行RNA-seq时候发现建库条带大小偶尔会偏小,建库质量较差。所以想了解下大家一般情况28S条带亮度能够达到多少,什么样的情况下,质量足以进行RNA-seq~

3/14/2021 6:56:39 PM Reply


Nan Liu
Chinese Academy of Sciences, China

关于RNA提取及建库质控的问题 我介绍我组钟叶丹,他是果蝇RNA抽提和建库specialist。钟叶丹的电话(同微信):13661616910。一般我们不用琼脂糖胶来判断果蝇RNA质量,果蝇total RNA(因为存在切割)是非典型胶图。可以咨询叶丹他质控的方式。

3/14/2021 7:03:40 PM Reply


鼎 裘
IBP

非常感谢~

3/14/2021 7:14:15 PM Reply


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