JHU Oncology Microarray Facility
RNA Preparation Protocol and Submission Guideline
Core uses and recommends TRIzol Reagent (Invitrogen) and RNeasy Mini Kit (Qiagen) for
RNA preparation. Look in kit’s handbook for details.
Tissue samples
1. Cut up 25 – 50 mg tissue into small pieces under frozen condition.
2. Without thawing the tissue, homogenize with 1 ml TRIzol Reagent in a 2 ml tube until
the tissue becomes uniformly homogeneous (usually take 20 – 40 sec).
3. Leave homogenate for 5 min at room temperature.
4. Add 200 l of chloroform to the tube and vortex for 15 sec.
5. Leave homogenate for 2 min at room temperature.
6. Transfer the sample into a Phase Lock Gel Heavy tube (5Prime, yellow-color tube,
spin 20 sec before use).
7. Centrifuge at 12,000g for 10 min at 4ºC to separate phases.
8. Transfer upper aqueous phase to a new tube and measure the volume.
9. Add 1 volume of 70% ethanol and mix well by pipetting. Do not centrifuge.
10. Pipett up to 700 l sample including any precipitate into an RNeasy mini column and
follow RNeasy Mini Kit (Qiagen) procedure for RNA isolation.
11. Follow RNeasy Mini Kit procedure for on-column RNase-free DNase treatment.
Cultured cells
Start with 5x 105 to 2x 106 cells in a culture dish.
Rinse the cells in culture dish with cold 1x PBS and suck PBS away.
Add 1 ml TRIzol Reagent to the plate and detach cells by a scraper or pipetting.
Transfer the sample to a tube and vortex for 1 min.
Leave the tube for 5 min at room temperature.
Add 200 l of chloroform to the tube and vortex for 15 sec.
Leave the tube for 2 min at room temperature
Transfer sample into a Phase Lock Gel Heavy tube (5Prime, yellow-color tube, spin 20
sec before use).
9. Centrifuge at 12,000g for 10 min at 4ºC to separate phases.
10. Transfer upper aqueous phase to a new tube and measure the volume.
11. Add 1 volume of 70% ethanol and mix well by pipetting. Do not centrifuge.
12. Pipette up to 700 l sample including any precipitate into an RNeasy mini column and
follow RNeasy Mini Kit (Qiagen) procedure for RNA isolation.
13. Follow RNeasy Mini Kit procedure for on-column RNase-free DNase treatment.
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Sample preparation and submission guideline
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February 2012
JHU Oncology Microarray Facility
RNA quantification and submission
Measure RNA concentration with UV spectrometer or NanoDrop.
Purified RNA should show OD260/280 between 1.8 – 2.2 and OD260/230 close to 2.0.
Dilute RNA to 75 – 100 ng/l and submit 1 – 2 g RNA to the Core.
If sample number is larger than 8, submit samples in 8-strip tubes or microplate.
Submit samples frozen with dry ice.
Fill service request form and sample sheet downloadable from Core’s website,
http://microarray.onc.jhmi.edu.
7. Send electronic version of sample sheet to the Core.
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Sample preparation and submission guideline
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February 2012