Ronna Hertzano

Sigma – Tri Reagent (T9424) – protocol

Take with you also –

-  Chloroform

-  Isopropanolol

-  75% ethanol

-  DEPC (diethylpyrocarbonate-treated water)

Final preparation of RNA should have 260/280 ratio of >1.7.

Typical yields –

Liver, spleen 6-10μg per 1mg tissue.

Kidney 3-4μg per 1mg tissue.

Skeletal muscle, brain 1-1.5μg per 1mg tissue.

Placenta 1-4μg per 1mg tissue.

Too low volumes of TRI REAGENT may cause a DNA contamination!

Steps in 'grey' are intended to reduce DNA contamination of samples.

Formal amount / Amount for experiment
1 / Homogenize tissue samples in TRI REAGENT.
The volume of tissue should not exceed 10% of the volume of TRI REAGENT. / 1ml per 50-100mg of tissue
2 / Centrifuge homogenate at 12,000g for 10min at 40C
If samples had a high fat content – there will be a layer of material on the surface to be removed.
The supernatant contains RNA and Proteins.
Transfer the clear supernatant to a fresh tube. / 10 min at 40C and then transfer
3 / Allow samples to stand for 5min at room temperature.
Goal: ensure complete dissociation of nucleoprotein complexes. / 5 min at RT
4 / Add 0.2ml of chloroform per ml of TRI REAGENT, cover sample tightly and shake vigorously for 15 seconds.
Cover sample tightly and shake vigorously for 15 seconds.
Allow to stand 2-15min at room temperature.
Centrifuge at 12,000g for 15min at 40C.
Sample should separate to three phases. / 0.2ml per 1ml of TRI REAGENT
shake 15 seconds
stand 2-15min RT
15min 12,000g 40C
5 / Transfer aqueous phase to a fresh tube and mix aqueous phase with 1/10 volume of isopropanolol, store samples at room temperature for 5min, and centrifuge at 12,000g for 10min at 40C. / 1/10 per 1ml of TRI REAGENT
stand 5min in RT
10min 12,000g 40C
6 / Transfer aqueous phase to a fresh tube and add 0.5ml of isopropanolol per ml of TRI REAGENT
Allow sample to stand 5-10minutes in room temperature.
Centrifuge at 12,000g for 10min at 40C. The RNA precipitate will form a pellet on the bottom of the tube. / 0.5ml per 1ml of TRI REAGENT
stand 5-10min in RT
10min 12,000g 40C
7 / Remove the supernatant and wash the RNA pellet by adding 1ml (minimum) of 75% ethanol per 1ml of TRI REAGENT used.
Vortex the sample and then centrifuge at 7,500g for 5min ar 40C
If RNA pellet floats – perform the wash in 75% ethanol at 12,000g. Samples can be stored in ethanol at 40C for at least 1 week and up to 1 year at (-20)0C. / Remove supernatant
1ml Ethanol 75% per 1ml TRI REAGENT
Vortex
7,500g 5min 40C
8 / Briefly dry the RNA pellet for 5-10min by air drying or under a vacuum. Do not let the RNA pellet dry completely – it will decrease solubility.
Add water and repeat mix by pipetting at 55-600C 10-15min. / Dry 5-10 by air
Add water.
Mix with micropipette at 55-600C 10-15min.