JHU Oncology Microarray Facility
MicroRNA Preparation Protocol and Submission
Core is able to isolate microRNA from different types of samples for our customers. Consult
core before you isolate your samples.
Core recommends using miRNeasy Mini Kit (Qiagen, cat. #217004) for microRNA preparation. The
following procedures are adopted from kit’s handbook. Consult handbook for additional details.
Frozen tissue samples
1. Place 5 – 25 mg frozen tissue into 1.5 ml Eppendorf tube (cut to smaller pieces under frozen
condition prior to this step if desired). Add 100 – 300 ul QIAzol Reagent.
2. Homogenize with Kontes Pestle Cordless Motor (Fisher Scientific) until the tissue becomes
uniformly homogeneous.
3. Add QIAzol to 700 ul and mix.
4. Leave homogenate for 5 min at room temperature.
5. Add 140 l of chloroform to the tube and vortex for 15 sec.
6. Leave homogenate for 2 min at room temperature.
7. Transfer the sample into a Phase Lock Gel Heavy tube (5Prime, yellow-color tube, spin 20 sec
prior to use).
8. Centrifuge at 12,000g for 10 min at 4ºC to separate phases.
9. Transfer upper aqueous phase to a new tube and measure the volume.
10. Add 1.5 volumes of 100% ethanol and mix well by pipetting. Do not centrifuge.
11. Pipette up to 700 l sample including any precipitate into an RNeasy mini column and follow
miRNeasy Mini Kit procedure for RNA isolation.
12. Follow miRNeasy Mini Kit procedure for on-column RNase-free DNase treatment (optional).
Cultured or FACS-sored cells
Start with 1x 104 to 1x 106 cells in a culture dish.
Rinse cells once in culture dish with pre-cooled 1x PBS (optional).
Add 700 l QIAzol 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 140 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).
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 1.5 volumes of 100% ethanol and mix well by pipetting. Do not centrifuge.
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Sample preparation and submission guideline
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February 2012
JHU Oncology Microarray Facility
12. Pipette up to 700 l sample including any precipitate into an RNeasy mini column and follow
miRNeasy Mini Kit procedure for RNA isolation.
13. Follow miRNeasy Mini Kit procedure for on-column RNase-free DNase treatment (optional).
FFPE tissue sections
Use miRNeasy FFPE Kit (Qiagen, cat. #217504) for RNA isolation from FFPE tissue sections
by following procedures in kit’s handbook.
Blood samples
Use miRNeasy Serum/Plasma Kit (Qiagen, cat. #217184) for microRNA/small 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 25 – 50 ng/l and submit 100 – 500 ng 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