Table 2 Overview of the RNA extraction protocols. Four RNA extraction protocols are Ambion RecoverAll™ Total Nucleic Acid Isolation Kit, Stratagene Absolutely RNA® FFPE Kit, Gentra Purescript® RNA Purification Kit and Invitrogen Trizol® Reagent. (RT. = Room temperature)
Ambion | Stratagene | Gentra | Trizol | |
|---|---|---|---|---|
De-paraffiniztion | Xylene 50°C for 3 min Ethanol washes | Deparaffin – Reagent RT. for 10 min Ethanol washes | Xylene RT. for 5 min Ethanol washes | Xylene RT. for 5 min Ethanol washes |
Proteinase K digestion | 4 μl Protease in 400 μl digestion buffer (final concentration unknown) 50°C for 3 hours | 10 μl × 20 mg/ml proteinaseK in 100 μl digestion buffer (final concentration 1.82 μg/ul) 55°C overnight | 1.5 μl × 20 mg/ml proteinaseK in 300 μl cell lysis solution (final concentration 0.10 μg/ul) 55°C overnight | -- |
RNA isolation | Add Isolation Additive Add Ethanol Pass the mixture through a Filter Cartridge Buffer washes On column DNase digestion | Add β-ME Pass the mixture through a prefilter spin cup Add Ethanol Pass the mixture through a RNA binding cup Buffer washes On column DNase digestion | Add protein-DNA precipitation solution Separate the supernatant Add Isopropanol and Glycogen -20°C for 1 hour Pellet RNA and Ethanol wash | Homogenize in 1 ml Trizol Reagent Add Chloroform Separate the aqueous phase Add Isopropanol and Glycogen RT. for 10 min Pellet RNA and Ethanol wash |
RNA collection | Elution buffer 30 μl × 2 | Elution buffer 30 μl | RNA hydration solution 30 μl | H2O 30 μl |