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LIRA reagent for RNA isolation

Features: The universal reagent  for RNA isolation from cells and tissue samples of animal and plant origin.
Precautionary measures:
Caution! The reagent for cell and tissue lysis contains agents that are toxic and cause irritation. Rules of general and personal safety must be abided when working with the reagent. The reagent is toxic when in contact with skin and if swallowed, causes burns.
Work in a fume hood! After contact with skin, wash immediately with a large amount of water and detergent. Seek medical attention, if necessary.
 
Product description:
The reagent is developed for RNA isolation from cells and tissue samples of animal and plant origin.
Protocol of use:
For RNA isolation from cells and tissues, it is necessary to carry out complete lysis of the biological sample with the use of "Lira" reagent. In order to do this, add the reagent to a sample in an amount sufficient to obtain transparent homogeneous lysate.
Approximate amounts of the reagent needed:
-        for mammalian cells harvested from monolayer or suspension by centrifugation – 900 µl per 2-5•103 cells;
-        in case of lysis in culture plates – 900 µl per 10 cm2 (reagent should evenly cover the entire area);
-        for tissue samples – 900 µl per 40-80 mg of tissue (the quality of RNA sample primarily depends on the conditions of tissue sample storage; tissue homogenization should be carried out directly in the reagent solution).
Carry out lysis at room temperature for 3-10 min. Collect lysate in 1.5 ml Eppendorf tube (standard tubes of larger volume can be used if necessary). Add chloroform to the lysate in the amount of 1/5 of the volume of the lytic reagent, stir vigorously for 15 seconds and divide phases by centrifugation at 10,000 g, 4 °C for 10 minutes. Collect aqueous phase gently (!!! avoid capturing interphase) and transfer it into a clean tube.
Add an equal volume of isopropyl alcohol to the aqueous phase, mix thoroughly and incubate at room temperature for 10 minutes (in order to increase the product yield, RNA can be kept at lower temperatures up to -20 °C and cold isopropyl alcohol can be used), then centrifuge at 16,000 g, 4 °C for 15 minutes. Remove the supernatant, rinse the pellet with 80% ethanol (volume of ethyl alcohol should comprise not less than half of the original lysate volume). For sediment formation on the tube bottom, centrifuge at 12,000 g, 4 °C for 5 minutes. The resulting precipitate must be air-dried for 15-30 minutes and then dissolved in deionized water (!!! RNase-free) for further use and storage.
After RNA isolation, meausure the absorbance of solutions at 260 and 280 nm wavelengths; 260/280 ratio for RNA solutions should correspond to the range of 1.60 – 1.95.
Notes:
-        when working with RNA, one should wear gloves and specially prepared RNase-free equipment and solutions in order to avoid RNA degradation;
-        in order to increase RNA yield, sodium acetate (up to 0.3 M) and coprecipitants (e.g., glycogen at a concentration of 5-10 mg/ml) can be used at the stage of precipitation.