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 1. 2. 3. 4. 5. 6. 7. 8. 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). ______________________________________________________________________________ Sample preparation and submission guideline Page 1 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 1. 2. 3. 4. 5. 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). ______________________________________________________________________________ Sample preparation and submission guideline Page 2 February 2012