TRIzol Reagent

RNA isolation / purification Cells - primary human dermal fibroblasts

Experiment
RNA isolation / purification Cells - primary human dermal fibroblasts
Product
TRIzol Reagent from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

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.

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Discussion

Discussion

5 years ago

Author: Switzerland

I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?

I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?

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Papers

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Manufacturer protocol

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

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