Jacobs:Protocol Total RNA Isolation using Tri Reagent
Revision as of 19:36, 16 June 2008 by Ashley Chou (New page: ==Materials== * Tri-Reagent (Sigma T9424) * Cell scraper * Pipetman * Pipet tips * Autoclaved microcentrifuge tube * Chloroform (Sigma (C2432) * Centrifuge * Isopropanol (Sigma I9516) * 75...)
- Tri-Reagent (Sigma T9424)
- Cell scraper
- Pipet tips
- Autoclaved microcentrifuge tube
- Chloroform (Sigma (C2432)
- Isopropanol (Sigma I9516)
- 75% Ethanol
- RNase free H2O
- Lyse cells directly on culture dish. Add 1 ml of TRI REAGENT (for up to 5x106 cells). Use cell scraper to scrape off cells and pass cell lysate through pipette 20 times to form homogeneous lysate. (Perform Steps 1-2 in fume hood)Note: If using plastic dish, work as quickly as possible since TRI REAGENT is not compatible with plastic.
- Transfer lysate to 1.5 ml microcentrifuge tube.
- Allow samples to stand for 7 mins at room temperature for complete dissociation of nucleoprotein complexes.
- Add 0.2 ml of chloroform. Cover the sample, vortex for 10 secs and allow to stand for 10 mins at room temperature. Centrifuge the resulting mixture at 12,000g for 15 mins at 4C. This separates the mixture into 3 phases: a red organic phase (protein) in the bottom, an interphase pellet (DNA) in the middle, and a colorless aqueous phase (RNA) at the top.
- Transfer top aqueous phase to a new autoclaved microcentrifuge tube and add 0.5 ml of isopropanol. Mix well and allow sample to stand for 10 mins at room temperature. Note: Store the interphase and organic phase at 4C for subsequent isolation of DNA and proteins.
- Centrifuge at 12,000g for 10 mins at 4C. The RNA precipitate will form a pellet on the bottom of the tube.
- Remove the supernatant and wash RNA pellet by filling tube with 75% ethanol (~1.5 ml). Vortex sample and then centrifuge at 7,500g for 5 mins at 4C. Note: Samples can be stored in ethanol at 4C for 1 wk and at -20C for 1 year.
- Pour out ethanol (be careful not to pour out RNA pellet). Place tubes upside down on Kimwipe to rid of trace of ethanol. Do not let the RNA pellet dry completely.
- Add 20 μL RNase free H2O to RNA pellet. Place tubes on ice. Resuspend pellet and transfer 1 μL into new tube with 99 μL RNase free H2O for RNA quantification.
- Turn on spectrophotometer (switch is on back of machine) let it self calibrate (~3 mins)
- Turn on UV lamp (takes ~ 10 mins): Function – Setup – Lamps – UV and Visible (325 nm)
- Wash cuvette with H2O.
- Mode – Nucleic – RNA – Background ON. Then reference with 100 l H2O.
- Remove H2O.
- Transfer sample in cuvette (100 l from Step 5) and run.
- Write down ratio and absorbance at 260, 280.
- Concentration (g/ml) = [(A260-A280)x100x40]
- Total Yield (g) = Conc x 0.019
- 260 to 280 ratio should be greater than 1.7.
- Turn off power.
- Store RNA at -20°C.
- Originally prepared by CRJ-CHK 2/17/04
or instead, discuss this protocol.