How do you isolate RNA with TRIzol?
Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization. Incubate samples at 15 to 30oC for 10 minutes and centrifuge at not more than 12,000 x g for 10 minutes at 2 to 4oC.
Why TRIzol is used in RNA isolation?
TRIzol™ Reagent maintains the integrity of the RNA due to highly effective inhibition of RNase activity while disrupting cells and dissolving cell components during sample homogenization.
How does TRIzol inhibit RNase?
TRIzol® Reagent maintains the integrity of the RNA, while disrupting cells and dissolving cell components, and also provides an immediate and highly effective inhibition of RNase activity during sample homogenization or lysis.
Does TRIzol denature RNA?
When Acidic, as is the case with TRIZOL, the double stranded nuclear DNA precipitates and ends up mostly in the interface. The phenol is an organic solvent and the guanidine isothiocyanate is a chaotropic agent that denatures proteins. When all of these are present the RNA is safe from degradation.
How much TRIzol do I add?
a. Add 1 ml of TRIzol or TRI reagent to every 50-100 mg of tissue. Sample volume should not exceed 100 μl. If you using 1.5 -2 ml microcentrifuge tube and pestle for homogenization, start with 500 μl of TRIzol or TRI reagent, then add remaining 500 μl.
How long is RNA stable in TRIzol?
Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol™ Reagent used for lysis. Note: The RNA can be stored in 75% ethanol for at least 1 year at –20°C, or at least 1 week at 4°C.
How long can cells stay in TRIzol?
If the biological sample is efficiently lysed in TRIzol and the reagent can inactivate the nucleases, RNA can be safely stored for 3 or 4 days at room temperature (20-25ºC).
Can you freeze RNA in TRIzol?
They can be stored indefinitely in a -80°C freezer until needed for RNA. Avoid allowing the sample to thaw prior to extraction. If dealing with a large piece of tissue, it can be stored in pieces to prevent multiple freeze-thaw cycles. TRIzol reagent will also work with cell pellets, which must also be stored at -80°C.
Can you use too much TRIzol?
Tissue. As a rule of thumb, the sample size should not be greater than 10% of the total volume of TRIzol used for lysis.