JHU Oncology Microarray Facility
RNA Preparation Protocol and Submission
Core is able to isolate RNA from different types of samples for our customers. Consult core
before you isolate your samples.
Core recommends using TRIzol Reagent (Life Technologies, cat. #15596026) and RNeasy Mini Kit
(Qiagen, cat. #74104) or RNeasy Micro Kit (Qiagen, cat. #74004) for RNA preparation. The following
procedures are adopted from kit’s handbook. Consult handbook for additional details.
Frozen tissue samples
1. Place 10 - 25 mg frozen tissue into 50 ml conical tube and add 1ml TRIzol (cut tissue into
smaller pieces under frozen condition prior to this step if desired).
2. Homogenize using a tissue homogenizer (PowerGen 125 homogenizer, Fisher Scientific) for
20 – 40 sec until the tissue becomes uniformly homogeneous.
3. For small tissue samples, place frozen tissue into 1.5 ml Eppendorf tube. Add 100 – 300 ul
TRIzol Reagent.
4. Homogenize with Kontes Pestle Cordless Motor (Fisher Scientific) until the tissue becomes
uniformly homogeneous. Add TRIzol to 1 ml.
5. Leave homogenate for 5 min at room temperature.
6. Add 200 l chloroform to the tube and vortex for 15 sec.
7. Leave homogenate for 2 min at room temperature.
8. Transfer the sample into a Phase Lock Gel Heavy tube (5Prime, yellow-color tube, spin 20 sec
prior to 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 sample including any precipitate into an RNeasy mini column and follow RNeasy Mini
Kit procedure for RNA isolation.
13. Follow RNeasy Mini Kit procedure for on-column RNase-free DNase treatment (optional).
Cultured or FACS-sorted cells
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Start with 1x 104 to 1x 106 cells in a culture dish.
Rinse attached cells once with pre-cooled 1x PBS (optional).
Add 1 ml TRIzol (or QIAzol) to the plate and detach cells by a scraper or pipetting.
Transfer cells to a tube and vortex for 1 min.
Leave the tube for 5 min at room temperature.
Add 200 l 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
prior to use).
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Sample preparation and submission guideline
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February 2012
JHU Oncology Microarray Facility
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. Transfer the sample onto RNeasy mini column and follow RNeasy Mini Kit procedures for
RNA isolation
13. Use RNeasy Micro Kit and follow its procedures if started with very small number of cells.
14. Follow RNeasy Mini Kit (or RNeasy Micro Kit) procedures for on-column RNase-free DNase
treatment (optional).
FFPE tissue sections
Use RNeasy FFPE Kit (Qiagen, cat. #73504) for RNA isolation from FFPE tissue sections by
following procedures in kit’s handbook.
Blood samples
Use PAXgene Blood RNA Kit (Qiagen, cat. #762164) for RNA isolation from blood samples
by following procedures in kit’s handbook.
RNA quantification and submission
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Quantify 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 20 – 100 ng/l and submit 50 ng – 1.0 g RNA to the Core.
Submit samples frozen in dry ice.
Submit service request via https://skcccjhmi.corefacilities.org/account/login. (Need to
download sample sheet when make the request and upload after filling in sample information).
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Sample preparation and submission guideline
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February 2012