RNA extraction: TRIzol procedure for cell lines followed by DNase and RNeasyNote:
This protocol is used to prepare RNA that will be used for for microarray hybridization or RT-PCR.
- Drosophila PBS: 2.7 mM KCl, 4.3 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, pH 7.2
- TRIzol® reagent: Invitrogen, cat. no. 15596-026 (100 ml) or 15596-018 (200 ml)
- DNase/RNase-free water: Invitrogen cat. no. 10977.
- 75% ethanol, made with DNase/RNase-free water.
- RNase-free DNase (Qiagen cat. no. 79254.)
- RNeasy® spin columns: RNeasy midi kit (Qiagen cat. no. 75142 or 75144).
- Pellet cells from 5-10 plates by centrifugation in 50 ml disposable polypropylene tubes (approximately 1000 x g, 5 min.).
- Discard the supernatant. Resuspend the pellet in 5 ml Drosophila PBS; transfer to a 15 ml polypropylene tube.
- Pellet cells again by centrifugation.
- Remove supernatant.
- Lyse the pellet in TRIzol reagent (0.75 ml per 5-10 x 107 cells) by repetitive pipetting. For 5 plates of cells at 5x106 cells/ml, use 2 ml TRIzol.
- Incubate at room temperature for 5 minutes.
- Add 0.267 ml chloroform per ml of TRIzol used in step 5.
- Shake tubes vigorously for 15 seconds.
- Incubate at room temperature for 2 minutes.
- Aliquot samples into 1.5 ml microfuge tubes.
- Centrifuge for 15 minutes at 4° at 12,000 x g.
- Transfer the top (aqueous) phases to clean tubes.
- Precipitate the RNA from the aqueous phase by adding 0.33 ml of isopropanol per ml of TRIzol used in step 5.
- Invert tubes once to mix gently.
- Incubate samples at room temperature for 10 minutes.
- Centrifuge for 10 minutes at 4° at 12,000 x g.
- Remove supernatant.
- Wash RNA pellet once with 75% ethanol, 0.7 ml per microfuge tube.
- Vortex briefly.
- Centrifuge at 7,500 x g for 5 minutes at 4°.
- Let pellet air dry for 10 minutes. Note: DO NOT let the pellet dry completely.
- Dissolve in RNase-free water. Yield should be around 1 mg from 10 plates of cells at 5 x 106 cells/ml; add enough water to give a final concentration around 2-5 mg/ml.
- Incubate at 37° overnight to dissolve the RNA. It may be stored at -80° at this point if desired.
- Purify up to 1 mg of RNA on an RNeasy spin column, with DNase treatment, precisely as described in Qiagen's RNeasy Midi/Maxi Handbook, supplied with the RNeasy kit; the handbook can also downloaded from http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000289. Do the optional second wash on the column. Elute twice, using 50 μl DNase/RNase-free water for each elution.
- The yield from the column is typically 60-80%. Determine concentration by absorbance, using a Nanodrop® ND-1000 Spectrophotometer. If necessary, the RNA can be concentrated on a SpeedVac. Store at -80°.
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