Based on the user guide of Trizol reagent by Invitrogen.

Material

Contents and storage
Contents Cat. No. 15596026 (100 reactions) Cat. No. 15596018 (200 reactions) Storage
TRIzolTM Reagent 100 mL 200 mL 15–30°C
Required items which is not supplied.
Item Memo
Equipment
Water bath or heat block at 55–60°C
Reagents
Isopropanol
Ethanol, 75%
RNase-free water of 0.5% SDS
(Optional) RNase-free glycogen

Input sample requirements

IMPORTANT!
Perform RNA isolation immediately after sample collection or quick-freeze samples immediately after collection and store at –80°C or in liquid nitrogen until RNA isolation.
Sample type Starting material per 1 mL of TRIzolTM Reagent
Tissues[1] 50 ー 100 mg of tissue
Cells grown in monolayer 1 × 10xx5 ー 1 × 10xx7 cells grown in monolayer in a 3.5–cm culture dish (10 cm2)
Cells grown in suspension 5 ー 10 × 10xx6 cells from animal, plant, or yeasty origin or 1 × 10xx7 cells of bacterial origin
[1] Fresh tissues or tissues stored in RNAlaterTM Stabilization Solution (Cat. No. AM7020).

Procedural guidelines

  • Perform all steps at room temperature (20–25°C) unless otherwise noted.
  • Use cold TRIzolTM Reagent if the starting material contains high levels of RNase, such as spleen or pancreas samples.
  • Use disposable, individually wrapped, sterile plastic ware and sterile, disposable RNase-free pipettes, pipette tips, and tubes.
  • Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin; change gloves frequently, particularly as the protocol progresses from crude extracts to more purified materials.
  • Always use proper microbiological aseptic techniques when working with RNA.
  • Use RNaseZapTM RNase Decontamination Solution (Cat.no. AM9780) to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.



Protocol

1 Lyse samples and separate phases
  • Lyse and homogenize samples in TRIzolTM Reagent according to your starting material.

Cell grown in monolayer:

  • Remove growth media.
  • Add 0.3–0.4 mL of TRIzolTM Reagent per 1 × 105—107 cells directly to the culture dish to lyse the cells.
  • Pipet the lysate up and down several times to homogenize.

Note: The sample volume should not exceed 10% of the volume of TRIzolTM Reagent used for lysis.

STOPPING POINT
Samples can be stored at 4°C overnight or at – 20°C for up to a year.
  • (Optional) If samples have a high fat content, centrifuge the lysate for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear supernatant to a new tube.
  • Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex.
  • Add 0.2 mL of chloroform per 1 mL of TRIzolTM Reagent used for lysis, then securely cap the tube.
  • Incubate for 2–3 minutes.
  • Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.
  • The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase.
  • Transfer the aqueous phase containing the RNA to a new tube. Transfer the aqueous phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.
  IMPORTANT! 
  Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase.   
  • Proceed directly to “Isolate RNA“



2 Isolate RNA

Precipitate the RNA

  • (Optional) If the starting sample is small (<106 cells or <10 mg of tissue), add 5–10 μg of RNase-free glycogen as a carrier to the aqueous phase.

Note: The glycogen is co-precipitated with the RNA, but does not interfere with subsequent applications.

  • Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzolTM Reagent used for lysis. Incubate for 10 minutes.
  • Centrifuge for 10 minutes at 12,000 × g at 4°C.
  • Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.
  • Discard the supernatant with a micropipettor.



Wash the RNA

  • Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzolTM Reagent used for lysis.

Note: The RNA can be stored in 75% ethanol for at least 1 year at –20°C, or at least 1 week at 4°C. Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C.

  • Discard the supernatant with a micropipettor.
  • Vacuum or air dry the RNA pellet for 5–10 minutes.
IMPORTANT! 
Do not dry the pellet by vacuum centrifuge. Do not let the RNA pellet dry, to ensure total solubilization of the RNA. Partially dissolved RNA samples have an A230/280 ratio <1.6.



Solubilize the RNA

  • Resuspend the pellet in 20–50 μL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting up and down.
  IMPORTANT! 
  Do not dissolve the RNA in 0.5% SDS if the RNA is to be used in subsequent enzymatic reactions.
  • Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.



Proceed to downstream applications, or store the RNA at –70°C.




Dan Yamamoto-Evans 2019/12/05 11:39

  • interactome/trizol.txt
  • Last modified: 2019/12/05 11:51
  • by dan