实验概要
This method is designed for most animal tissues and culture cells. For RNA isolation from fibrous tissue, follow the specialized protocol on page 11. For laser dissected samples, please follow the protocol on page 5. All centrifugation step must be carried out at room temperature.
主要试剂
Regents supplied by user:
1. 96-100% ethanol
2. $-Mercaptoethanol
主要设备
Equipments supplied by user:
1. RNase-free filter pipette tips
2. Microcentrifuge capable of 12,000 x g
实验步骤
1. Determine the starting amount of sample. Do not use more than 5 x 105 cells or 5 mg tissue.
2. Lyse cells (< 5x 105) or tissues (< 5mg) with 350 ul of TRK Lysis Buffer. Remember to add 20 ul of 2-mercaptoethanol per 1 mL of TRK Buffer before use.
3. Disrupt the tissue or cells and Homogenize the lysate in TRK Lysis Buffer according to one of the methods on page 4. When processing small amounts of cells (.5000 cells). Add 3ul of linear acrylamide and 1ul Carrier RNA to the lysate before homogenization.
4. Centrifuge at 13,000 x g for 3 minutes at room temperature when processing animal tissue.
5. Transfer the supernatant to a new 1.5 ml centrifuge tube and add equal volume of 70% ethanol to the lysate. Mix thoroughly by vortexing or pipetting.
6. Apply the mixture from step 5 onto HiBind® MicroElute® RNA column preinserted in a 2 mL collection tube. The maximum capacity of the spin cartridge is 750 ul. A precipitate may form on addition of ethanol in step 5. Vortex and add the entire mixture to the column. With the spin column inside a 2ml collection tube (supplied with kit), centrifuge at 10,000 x g for 30 seconds at room temperature. Discard flow-through and the collection tube.
7. Place column in a new 2 ml collection tube (supplied), and add 400 ul RWC Wash Bufffer. Centrifuge and discard flow-through. Reuse the collection tube in step 8 or step 9. If on-membrane DNase I digestion is desired, proceed step 8, otherwise go to step 9.
8. DNase I Digestion (Optional): this is point to start On-membrane DNase I digestion. (See detail procedure on page 14).
9. Place column in the same 2ml collection tube and add 500 ul RWB Wash Buffer diluted with absolute ethanol. Centrifuge at 10,000 x g for 30 seconds. Discard the flow-through and re-use the collection tube.
Note: RWB Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for instructions
10. Wash column with a second 500 ul of RWB Wash Buffer as in step 9. Centrifuge at 10,000 x g and discard flow-through. Then with the collection tube empty, centrifuge the column for 2 min at full speed ($13,000 x g) to completely dry the HiBind® matrix.
11. Elution of RNA. Transfer the column to a clean 1.5 mL microfuge tube (not supplied with kit) and elute the RNA with 15-20 ul of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. Let it sit at room temperature for 2 minutes. Centrifuge 1 min at maximum speed.
RNA may be eluted with a smaller (<15ul) volume of water to get higher RNA concentration. While reduced elution volume decrease total RNA yield. The total yield will be 20-30% less when the elution volume is less than 10ul.
RNA编辑技术通过改变RNA序列来“补偿”有害的突变,使正常蛋白得以合成。RNA编辑也可增加有益蛋白的产生。与CRISPR基因组编辑不同,RNA编辑不会改变基因,也不会产生永久性的变化。图片来源:视觉......
植物是复杂的生物系统。植物体内基因的表达受到多种水平的调控,如转录水平、转录后水平、DNA甲基化/去甲基化等,从而对基因表达进行精密高效的调控。中国科学院遗传与发育生物学研究所张劲松研究组筛选OsEI......
博德研究所张锋团队在PNAS 在线发表题为“HumanparaneoplasticantigenMa2(PNMA2)formsicosahedralcapsidsthatcanbeengin......
今年1月9日,我国继美国、日本之后,正式批准治疗阿尔茨海默病的新药仑卡奈单抗上市。这款药曾被美国《科学》杂志列为2023年度十大科学突破之一。如何攻克阿尔茨海默病一直是医学界的重要课题。据世界卫生组织......
日,《科学通报》在线发表了华南农业大学园艺学院教授夏瑞团队最新研究成果,他们研究开发出一款多功能植物小RNA分析工具——sRNAminer,可便于研究人员进行一站式小RNA分析及可视化。据介绍,植物小......
RNA干扰是指由双链RNA诱导的基因沉默现象,在细胞发育和抗病毒免疫等生物学过程中发挥重要作用,并被用作基因功能研究和疾病治疗的遗传工具。RNA干扰现象可在秀丽隐杆线虫全身及其后代中传播,被称为系统性......
生物体的复杂性是由它们的基因编码的,但这些基因从何而来?据最新一期《美国国家科学院院刊》报道,芬兰赫尔辛基大学研究人员解决了围绕小分子RNA基因(microRNA)起源的悬而未决的问题,并描述了一种创......
11月30日,中国科学院生物物理研究所叶克穷课题组在《中国科学:生命科学(英文版)》(ScienceChinaLifeSciences)上,在线发表了题为Complicatedtargetrecogn......
近日,中国科学院水生生物研究所张承才团队关于细菌中RNA代谢调控机制的研究取得了进展。相关研究成果以《蓝藻中RNaseE受一个保守蛋白调控》(Aconservedproteininhibitorbri......
应激颗粒是在胁迫条件下形成的动态结构,通常认为其中包含翻译被抑制的RNA以及翻译元件,并可在刺激消失后解聚,是细胞内典型的无膜细胞器。在应激颗粒组装的不同阶段,大量RNA分子会被招募至应激颗粒中,对维......