RNA提取试剂 （TRIZOL）
Description:TRIZOL is a mono-phasic reagent for the isolation of total RNA from cells and tissues. The reagent, containing phenol and guanidine isothiocyanate, extracts total RNA during lysing the cells in a single-step procedure.
Procedure:
I. RNA Preparation for Cells Grown in Monolayer
1. Lyse cells directly in a culture plate/dish/flask by adding 1 ml of TRIZOL Reagent to a 3.5-10 cm dish or similar cell culture devices, and passing the cell lysate several times through a pipette.
2. Incubate the homogenized samples for 5 minutes at room temperature (RT) to permit the complete dissociation of nucleoprotein complexes.
3. Add 0.2 mL of chloroform per 1 mL of TRIZOL Reagent. Shake tubes vigorously for 15 seconds and incubate them at RT for 3 minutes. (see note 1)
4. Centrifuge the samples at 10,000 × g for 10 minutes at RT.
5. Transfer the aqueous phase to a fresh tube. 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.
6. Incubate samples at RT for 10 minutes and centrifuge at no more than 10,000 × g for 10 minutes at 2 to 8°C.
7. Remove the supernatant. Wash the RNA pellet once with 75% ethanol.
8. Centrifuge at 10,000 × g for 5 minutes at 4°C.
9. Briefly dry the RNA pellet (see note 2). Dissolve RNA in RNase-free water.
I. RNA Preparation for Cells Grown in Suspension
1. Pellet cells by centrifugation.
2. Lyse cells in TRIZOL Reagent by repetitive pipetting.
3. Continue I) step 2-9.
III. RNA Preparation for Tissues
1. Homogenize tissue samples in 1 mL of TRIZOL Reagent per 50-100 mg of tissue using a glass-Teflon or power homogenizer.
2. Continue I) step 2-9
Notes:
1. DO NOT vortex the samples, which will cause DNA contamination.
2. DO NOT over-dry the RNA, which will decrease its solubility.
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