[Bio101] RNA Preparation for Microarray Experiments

Materials and Reagents

1. S. pombe cells
2. Qiagen RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
3. DNase I (Roche Diagnostics, catalog number: 04716728001)
4. RNA STAT based on "S. Pombe RNA Prep"
5. DEPC water
6. EDTA
7. RLT buffer
   

Equipment

1. Standard bench-top centrifuge with 1.5 ml Eppendorf tube capacity
2. Calibrated standard laboratory pipettes
   

Procedure

1. Total RNA extraction
   Grow 5-10 OD log phase S. pombe cells, extract total RNA using RNA STAT based on "S. Pombe RNA Prep".
   Note: Using DEPC water to dissolve RNA. Usually RNA concentration is > 1.2 μg/μl and A₂₆₀/A₂₈₀=1.95-2.15.
   
2. DNase I treatment
   Prepare the following mixture:
   

   Total RNA	50 μg
   10x incubation buffer	5 μl
   DNase I	1 μl (10 units)
   Water (RNase free)	up to 50 μl
   Total	50 μl

   Incubate at room temperature (RT) for 20 min, then stop the reaction by adding 2 μl of 0.2 M EDTA (pH 8.0) to a final concentration of 8 mM and heating to 75 °C for 10 min.
   Notes:   
   
   1. If RNA samples are not prepared right before the DNase I treatment, it is better to re-measure RNA concentrations.
   2. Digestion time should not be more than 20 min. Higher temperatures and longer time could lead to Mg²⁺-dependent hydrolysis of RNA. Additionally, it is vital that EDTA be added to at least 2 mM prior to heat-inactivation to avoid this problem.
   3. RNA cleanup is strongly recommended right after DNase I digestion. If you could finish step 1 of RNA cleanup (add RLT and mix well) within 1 min, DNase I inactivation step is not necessary.
      
3. RNA cleanup (Qiagen RNeasy Mini Kit)
   1. Before RNA cleanup, make sure:
      1. There is no precipitate in RLT buffer.
      2. Buffer RPE is ready to use (EtOH added).
      3. All reagents and the centrifuge are at RT 20-25 °C. 
      4. Enough columns, collection tubes are available.
         
   2. Adjust the sample to 100 μl with RNase-free water. Add 350 μl Buffer RLT and mix well.
   3. Add 250 μl EtOH, mix well by pipetting.
   4. Transfer the sample to a RNeasy Mini spin column placed in a 2 ml collection tube. Close the lid gently and centrifuge for 15 sec at 10,000 x g. Discard the flow-through.
   5. Add 500 μl buffer RPE to the column. Close the lid gently, and centrifuge for 15 sec at 10,000 x g. Discard the flow-through.
   6. Add 500 μl buffer RPE to the column. Close the lid gently, and centrifuge for 2 min at 10,000 x g. Carefully remove the column from the collection tube so that the column does not contact the flow-through.
   7. Place the column to a new collection tube (2 ml), close the lid gently and centrifuge for 1 min at 10,000 x g.
   8. Place the column in a new collection tube (1.5 ml). Add 30 μl RNase-free water directly to the spin column membrane. Close the lid gently and centrifuge for 1 min at 10,000 x g to elute the RNA.
   9. Add the eluate from STEP 7 back to the spin column membrane. Close the lid gently and centrifuge for 1 min at 10,000 x g to elute the RNA.
      Note: Work quickly!
      
4. Sample preparation for microarray
   
   1. Measure RNA concentrations.
   2. Check whether RNA samples are degraded (Northern blot, check rRNA).
   3. Optional: Analyse one negative control (tube 1 etc) and one positive control using northern blot or real-time RT-PCR.
   4. Adjust RNA concentration to 1.0 μg/μl with RNase-free water. Prepare 10 μg total RNA for microarray analysis.
      

Notes

1. All steps need to be conducted rapidly to minimize chances of RNA degradation. All materials and laboratory space used in the experiment needs to be kept clean to ensure maximum likelihood of undegraded RNA.

Acknowledgments

This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.

References

1. Manual for DNase I recombinant, RNase-free (Roche, Catalog number: 04716728001)
2. RNeasy Mini Handbook (p. 56-57)