TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

Fibroblasts (1×106) were combined with 1 mL TRIzol (Invitrogen, Carlsbad, CA, USA) reagent and homogenized. The homogenate was allowed to stand for 5 minutes. Then, 200 μL chloroform (per 1 mL TRIzol) were added, and the mixture was shaken vigorously for 15 seconds. The sample was allowed to stand for 3 minutes at room temperature and centrifuged at 12,000×g at 4°C for 15 minutes. The colorless, upper aqueous phase was transferred to a diethylpyrocarbonate (DEPC)-treated Eppendorf centrifuge (EP) tube. Next, 500 μL of 100% isopropanol and 1 mL TRIzol were added to the aqueous phase and inverted 30 times. After incubating for 10 minutes at room temperature, the mixture was centrifuged for 10 minutes at 12,000×g at 4°C. The isopropanol layer was decanted, and the RNA pellet was washed with 1 mL cold 75% DEPC-treated water/ethyl alcohol. The samples were centrifuged for 5 minutes at 7,500×g at 4°C, and the supernatant was decanted carefully. The pellet was allowed to air dry for 5 to 10 minutes at room temperature under a laminar flow hood without over drying. Then, 20 μL DEPC-treated H2O were added to the dried pellet and mixed with a pipette tip on ice. The RNA samples were warmed to 55 to 65°C in a heating block for 10 to 15 minutes. To assess the quantity and purity of the RNA, the optical density of the total RNA (diluted 1:100 with DEPC-treated H2O) was determined by spectrophotometry.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Cells - primary human dermal fibroblasts using TRIzol Reagent from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for TRIzol Reagent below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing RNA isolation / purification Cells - primary human dermal fibroblasts using TRIzol Reagent from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.