In this RNA isolation from tissue with trizol protocol post we have briefly explained about isolation of RNA principle, requirements, isolation of RNA procedure, and quantification of RNA.
Isolation of RNA
A biological sample is homogenised with the reagent using a glass homogeniser. After extraction with chloroform, the homogenate separates into two phases. RNA remains in the aqueous phase while DNA and proteins are extracted into the organic phase. The RNA is precipitated from the aqueous phase by addition of isopropanol, washed with ethanol and dissolved in RNase-free water.
Single step guanidium acid phenol method emphasizes on the ability of guanidium isothiocyanate (GITC) to lyse cells, denature protein and inactivate intracellular ribonuclease rapidly. The presence of β mercaptoethanol in the mixture increases the solubilisation properties of the GITC extraction buffer.
Acid phenol extraction (pH< 5.0) selectivity keeps cellular DNA in the organic phase and help in extraction of proteins and lipids. The addition of chloroform further removes lipids and produce two distinct phases containing the DNA, proteins and lipids and an aqueous phase containing the RNA.
4. Micro centrifuge Tubes
5. Micro centrifuge tube stand
TRIR kit has the following components: Phenol, guanidium isothiocyanate, urea, detergents, buffering agents and stabilizers.
1. Add 1 ml of TRIR to the homogenised tissue sample and swirl gently for 15 min and then keep at 4°C for 5 min to permit complete dissociation of nucleoprotein complexes.
2. To this, add 0.2 ml chloroform, shake vigorously for 15 sec and place on ice at 4°C for 5 min.
3. The lysate is to be then centrifuged at 12,000 x g for 15 min at 4°C to yield lower organic phase containing DNA and proteins and upper aqueous phase containing RNA.
4. The volume of the aqueous phase will be approximately 40-50% of the total volume of the lysate.
5. The aqueous phase to be carefully transferred to a fresh Eppendorf micro centrifuge tube without disturbing the interphase. Equal volume of isopropanol to be added, mixed and kept at 4°C for 10 min. It is to be again centrifuged at 12,000 x g for 15 min at 4°C to precipitate the RNA.
6. Discard the supernatant and wash the pellet twice with 75% ethanol and air dried. Dissolve the RNA pellet in 50ml of sterile milliQ water and place in a water bath at 60°C for 10 min to ensure maximum solubility of RNA. Vortex gently the RNA sample and quantify before storing at -80°C.
Diluted RNA sample to be quantified spectropotometrically by measuring the absorbance (A) at 260 nm. An absorbance of 1OD is equivalent to RNA concentration of 40 mg/ml. Therefore, the yield can be calculated by multiplying the absorbance at 260 nm with dilution factor and 40 mg. The purity of RNA preparations were assessed by determining the ratio of absorbance of sample at 260 nm and 280 nm.