Posted on May 18th, 2016 by UNM CC
v.2 M. Fero 6/1/06
- Homogenize cells (10 million) or tissue (50-100 mg) in 1 mL TRIzol Reagent (e.g. scrape and pass through 30G needle, dounce homogenize and pass through needle, or use a homogenizer) and transfer to a 1.5 mL tube.
Optional - Spin at 12k g for 10' at r.t. to pellet debris. Save pink supernatant in fresh tube.
- Incubate 5' at r.t.
- Add 0.2 mL CHCl3 (chloroform). Shake 15". Incubate 2-3' at r.t.
- Spin 12K g 15', 2-8ºC.
- Transfer clear (aqueous) phase to a fresh tube.
Optional - Add 5 - 10 µg glycogen if < 10 mg of tissue was used at start.
- Add 0.5 mL isopropanol, mix. Incubate at r.t. 10'. (Use 0.75 mL isopropanol for isolating miRNA)
- Spin 12K g 15', 2-8ºC.
- Discard supernatant. Add 1 mL 70% EtOH (with fresh with RNAse free H2O). Use 75% EtOH for miRNA. Vortex.
- Spin 7500 g 5', 2-8ºC. Optional: Repeat 75% EtOH wash.
- Discard supernatant. Air dry 5-10'.
- Dissolve RNA in RNAse free H2O.
See Fluorometer protocol for more information.
- Create a Ribogreen MasterMix: Turn on Fluorometer. Thaw 20x RNAse free TE, Ribogreen and RNA standard.
- Master Mix (multiply by # samples + 2 standards)
- 5 µL 20x T.E.
- 0.5 µL RiboGreen (200x)
- 92.5 µL DEPC treated H2O
- Aliquot 98.5 µL master mix into 1.5 mL tubes.
- Setup samples and controls:
- Add 2 µL H2O into one tube as the "blank".
- Add 2 µL of RNA standard into a tube as the "100 ng/uL" standard.
- Dilute each sample 1/10 in DEPC H2O (1 µL + 9 µL water).
- Add 2 µL dilute sample to the master mix aliquots.
- Blank and then calibrate the fluorometer using the "blank" and "100 ng/µL" standard.
- Measure each RNA sample. Multiply the results by 10x (dilution factor).
DNAse I Digestion
- Create DNAse I buffer: MgCl2 and KCl should directly treated with 0.1% DEPC, incubated o.n. at room temp. and then autoclaved. Create 1M Tris pH8.4 by adding 4.03 g Tris base + 2.64 g Tris HCl in 50 mL DEPC treated H2O. (Tris buffers can't be treated with DEPC). Sterile filter.
- Digest contaminating genomic DNA:
- 100 µg RNA
- 10 µL 10x DNAse I buffer (Tris pH8.4, 20 mM MgCl2, 500 mM KCl)
- 5 µL DNAse I (RNAse free)
- q.s. 100 µL with DEPC H2O
- Incubate at room temp. x10 min. Immediately proceed to next step.
RNeasy Mini Prep
(See RNeasy Mini Handbook for details)
- Add 0.6 mL of RLT solution.
- Transfer to a 1.5 mL tube.
- Add 1 vol (0.6 mL) 70% EtOH. Mix by pipetting. Transfer 0.6 mL to a minicolumn in a collection tube.
- Spin >10K rpm 15". Discard flow through. Return column to same tube.
- Load remainder of lysate to column. Spin, discard flow through.
- Wash by adding 0.7 mL RW1. Spin, discard flow through.
- Wash by adding 0.5 mL RPE. Spin, discard flow through.
- Repeat wash with 0.5 mL RPE, but spin for 2' to dry column.
(Be sure no buffer is carried over on column tip.)
- Elute by adding 50 µL RNAse-free H2O and transfer to a fresh tube. Spin >10K rpm 1'. Add another 50 µL H2O and spin again. Save pooled eluate at -20º or - 80ºC.
- Dilute ~50 ng of total RNA to 3µL with DEPC H2O, and submit to genomics lab.
- Check account for results. Non degraded RNA will have a 28s/18s ratio = 2. The "concentration" result may be equated to the amount of RNA (ng) that was submitted. For more details on the interpretation of BioAnalyzer tracings see the section of the BioAnalyzer Nano 6000 kit manual that discusses assay results.