TRIzol Products - IL (2024)

FAQs

How much TRIzol to use for RNA extraction? ›

TRIzol^® RNA Extraction Protocol with Direct-zol Miniprep

Remove growth media from culture dish, or collect cell suspension and centrifuge to discard the supernatant. Add 1 mL of TRIzol^® per 1 x 10⁵ – 1 x 10⁷ cells or 0.1 mL TRIzol^® per cm² of culture surface area.

The volume of TRIzol added should be as follows: For 100 mm plate use 1 ml TRIzol. For 1 well of a 6 well plate or a 35 mm plate use 500 µl TRIzol. For 1 well of a 24 well plate or a 15 mm plate use 200 µl TRIzol.

Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial hom*ogenization. Mix the sample by vortexing and centrifuge at no more than 7,500xg for 5 minutes at 2 to 8°C.

During tissue hom*ogenization or lysis, the TRIzol reagent maintains RNA integrity, while disrupting cells and dissolving cell components. Addition of chloroform followed by centrifugation separates the solution into an aqueous phase and an organic phase.

However, there are some drawbacks to using Trizol. One disadvantage is the low quality of extracted RNA due to contaminants from the residue of phenol and guanidinium thiocyanate from the reagents. This can affect downstream applications and the accuracy of results.

Always maintain a ratio of 3:1 between the volume of TRIzol™ LS Reagent and the sample. To facilitate isolation of RNA from small quantities of samples ( <106 cells or <10 mg of tissue) or for sample volumes <0.25 mL, adjust the sample volume to 0.25 mL with RNase-free water.

Trizol® is available as 100 and 200 ml solution in brown glass bottles. When stored at room temperature, it is stable for 12 months, but Invitrogen recommends storage at 2-8°C.

Sample hom*ogenization step: If samples are efficiently lysed in TRIzol® Reagent and the reagent inactivates the nucleases, you can safely store RNA for 3–4 days at room temperature.

 Do not vortex Trizol lysates as it can sheer RNA. (Vortexing is a different mechanical action than bead mill hom*ogenization.)

Remember that TRIzol can only inactivate RNases with which it is in direct contact—therefore tissue samples are not safe from RNA degradation until completely hom*ogenized.

Can I store tissue in TRIzol for RNA extraction? ›

Cannot store samples in TRIzol for long time, better you put your sample in RNA later then keep it -20 c for long time. RNA later prevents RNA degradation of the sample. If you want to hom*ogenize as soon as after collecting your samples then you transfer directly into the TRIzol.

Trizol is a highly corrosive and toxic chemical that can cause burns on contact with the skin as well as systemic poisoning.

The guanidinium salt serves as a chaotropic agent to denature proteins and the phenol (commonly indicated as a pink color) is an organic compound also used to extract nucleic acids and proteins ^3,4.

The name itself is derived from the method, since this method isolates the RNA, DNA, and proteins in three separate phases [9]. The RNA can be isolated using TRIzol as described in the following steps: (i)

We routinely freeze cell pellets in Trizol at -70 for extended periods of time with no noticeable differences in RNA yield compared with fresh samples. From the Trizol protocol: "hom*ogenized samples can be stored at room temperature for several hours, or at –60 to –70°C for at least one month."

Ideally we would like 5 ul of total RNA at a concentration of 100 -200ng/ul to perform TapeStation analysis and to confirm sample concentration and purity. Once the total RNA samples have passed these quality measures, the samples can be used for library preparation which requires approximately 300 ng of total RNA.

hom*ogenize tissue samples in 1 ml of TRIZOL reagent per 50 to 100 mg of tissue using a glass-Teflon or power hom*ogenizer (e.g. Polytron, Tekmar's TISSUEMIZER). The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for the hom*ogenization.

The A260/A280 ratio is used to assess RNA purity. An A260/A280 ratio of 1.8 2.1 is indicative of highly purified RNA. UV spectroscopy is the most widely used method to quantitate RNA. It is simple to perform, and UV spectrophotometers are available in most laboratories.

How much RNA is needed for RNA-seq? For standard RNA-seq library preparation, you should aim to start with between 100 ng to 1 µg of purified RNA. At least 500 ng purified total RNA is recommended by some core facilities.