Isolation of RNA from tissue samples was performed using a modification of the method described by Hansen, et al.[48 (link)]. Up to 200mg of tissue was disrupted in TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) with stainless steel beads (2x5 mm) using a TissueLyser (Qiagen, Germantown, MD) for three minutes at 25 r/s twice. Following homogenization, samples in TRIzol were separated using bromo-chloro-propane (Sigma, St. Louis, MO). The aqueous phase was collected into a new tube and glycogen was added as a carrier. The samples were washed in isopropanol and ethanol-precipitated overnight at -20°C. RNA was then fully re-suspended in 5 mM Tris pH 8.0.
Reagents used for RNA extraction (TRIzol Reagent, bromo-chloro-propane, isopropanol, ethanol)
dependent variables
RNA yield and quality
control variables
Tissue sample size (up to 200mg)
Stainless steel beads (2x5 mm) used for tissue disruption
TissueLyser settings (3 minutes at 25 r/s, twice)
Glycogen added as a carrier
Overnight precipitation at -20°C
Final RNA resuspension in 5 mM Tris pH 8.0
controls
Positive control: Not specified
Negative control: Not specified
Annotations
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