Question

How could you isolate mRNAs from other RNAs in the cell?

Answer 1

Answer:- Isolation of mRNA from cells is a difficult process and for this purpose different kits are used to isolate mRNA.Probes for mRNA and complementary RNA are used to isolate mRNA .The total RNA of a cell contains mRNA,tRNA,rRNA and other sequences which are non; coding . To seprate all of these RNA from mRNA cells are allowed to burst exposing their content.These cells are bursted with the help of Centrifugation with a compound TRIzol Reagent.

To isolate RNA content is formed as a tinted RED colored layer which is seprated out. Now mRNA is extracted out from this layer by various kits prepared such as INVITROGEN'S FAST TRACK 2.0 and another one is AMBION'SPOLY(A) isolation kit.

PROCEDURE:-

1) RNA content of total cell is mixed with RNAase buffer of the kit .

2) This mixture is heated at approx. 65 degree temp. and then instantly cooled on ice for a minute.

3)This mixture is mixed with NaCl and all oligo dT are completely dissolved.

4) This solution is subjected to centrifugation and then washed several times with buffer of the kit.

5) mRNA is eluted several times.

6)This elute is precipitated with the help of Sodium acetate and ethanol precipitation. this is then resuspended in Diethyl polycarbonate treated water.

7) this is stored at -80 degree temp. and analyses for quality and quantity by the process of Spectrophotometry.

Through these steps mRNA can be purified from other RNA of cell.