RNA Preparation for use on Bradfield Lab EDGE Microarrays

Revised May 2003

 

 

Tissue Preparation (Liver and Other Large Tissues)

 

  1. Tissues are harvested from animals, weighed if necessary, and immediately submerged in RNAlater solution (qiagen.com).
  2. The volume of RNAlater used is generally 10x the volume of tissue being preserved. All tissues larger than 500mg are snipped a few times to increase surface area.
  3. Tissues are stored at 4˚C for 24 hours. After the initial 24 hours, tissues are removed from RNAlater and 100-200mg of tissue is dissected away for RNA isolation. The remaining tissue is placed in 1.5ml tubes and stored at -80˚C.

 

Tissue Preparation (Fetal and Other Small Tissues)

 

  1. Tissue are harvested from animals, weighed if necessary, and immediately submerged in ten times the volume of RNAlater solution (qiagen.com).
  2. Tissues are stored at 4˚C for 24 hours. After the initial 24 hours, tissues are removed from RNAlater and 20mg of tissue is dissected away for RNA isolation. The remaining tissue is placed in 1.5ml tubes and stored at -80˚C.

 

Cell Preparation (Check QIagen Protcol to Optimize for Cell Type)

 

  1. Spin down 4x10^6 cells and wash twice with PBS.
  2. Resuspend cells in 350ul RLT with BME added.
  3. Homogenize cells for 30s with rotor-stator homogenizer.
  4. Freeze homogenate until ready for RNA isolation.

 

 

RNA Isolation

 

  1. Tissues are homogenized for 60 seconds using a rotor-stator homogenizer in the appropriate volume of RLT buffer with BME (see qiagen protocol).
  2. All remaining steps are followed using the Qiagen RNEasy Handbooks at http://www.qiagen.com/literature/index.asp.

 

RNA Concentration

 

  1. Total RNAs that do not meet concentration requirements are EtOH precipitated using RNase-free 3M sodium acetate pH=5.5 (1/10 volume) and 100% EtOH (2.5x volume). The precipitation is incubated at -30˚C for a minimum of two hours and then centrifuged at maximum speed for 30minutes. The pellet is washed with 70% EtOH, dried, and resuspended in an appropriate volume.

 

Quality Control

 

All total RNAs are tested for quality vi two methods:

 

  1. 5ul of total RNA is ready at A260 and A280, so that a ratio may be obtained. We accept total RNA with ratios from 1.8-2.1, although exceptions can be made if the RNA looks to be high quality on a gel.
  2. 5ul of total RNA is ran on a 1% agarose gel to check for degradation.