RNAIsolationFromAnimaltissueorcellculture

實驗概要

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.

主要試劑

Regents supplied by user:

1. 96-100% ethanol

2. $-Mercaptoethanol

主要設備

Equipments supplied by user:

1. RNase-free filter pipette tips

2. Microcentrifuge capable of 12,000 x g

實驗步驟

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.