Life Technologies
TRIzol
15596018
I used this product to extract RNA from peripheral blood mononuclear cells. Since the sample collection took quite a while, I saved my sample cells in the RNAlater solution. Initially, I tried the RLT buffer from Qiagen. But the RLT buffer seemed to not be strong enough to lysis the cells treated with RNAlater solution. I found TRIzol was much powerful at lysising cells.
For RNA extraction
Human peripheral blood mononuclear cell
1. Preparing Samples 1) Homogenizing samples (Add Trizol to unthawed sample directly) a. Add 1.0 mL of TRIzol to the sample. b. Lyse cells in sample by pipetting up and down several times 2) Phase separation a. Incubate the homogenized sample for 5 minutes at room temperature. b. Add 0.2 mL of chloroform. c. Shake tube vigorously by hand for 15 seconds. d. Incubate for 2–3 minutes at room temperature. e. Centrifuge the sample at 12,000 × g for 15 minutes at 4°C. f. Transfer the aqueous phase of the sample into a new tube. 2. RNA Isolation Procedure 1) RNA precipitation a. Add 0.5 mL of 100% IPA to the aqueous phase. Mix by gently inverting for 6 times. b. Incubate at room temperature for 10 minutes. c. Centrifuge at 12,000×g for 10 minutes at 4°C. 2) RNA wash a. Remove the supernatant from the tube. b. Wash the pellet, with 1 mL of 75% ethanol. c. Vortex the sample briefly, then centrifuge the tube at 7500×g for 5 minutes at 4°C. d. Invert the tube (opening down) and air-dry the RNA pellet for 10 minutes. 3) RNA suspension a. Resuspend the RNA pellet in RNase-free water (20-50 µL) by passing the solution up and down several times through a pipette tip. b. Incubate in a water bath set at 55°C for 10 minutes. Note: the volume for each reagent should be adjusted to the starting cell counts.
Wear PPE and perform in the fume hood, as TRIzol can be very dangerous!
If done with great care, a high yield of RNA can be obtained and the quality of RNA is usually good enough for various downstream applications.
Proceed to RNA cleanup if the quality of the RNA is critical.
Affordable, easy
Might require further cleanup, depending on downstream applications
It is a must for RNA extraction.
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