How can I purify total mRNA in animal cellsSolutionPurificat

How can I purify total mRNA in animal cells?

Purification of total mRNA in animal cells.

[Precaution: Always wear gloves. Treat your plasticware and glassware with DEPC O/N]

1. Cell lysis:

Lyse cells in one mL of Trizol or RNAzoi solution

2. Phenol: Chloroform isolation:

Add 0.2 mL of chloroform to the intial volume of the Trizol used (that is use 200microliters of Chloroform)

Mix vigorously for nearly 30 sec

Allow it to stand at room temperature to allow the phases to separate [Trizol already contains phenol]

Spin the tubes at 10,000g for 10 minutes at 4^oC

Carefully remove the upper clear layer of the chloroform containing RNA and collect it in a clear tube.

[Its wise to leave some chloroform in order not to disturb the lower pinkish phenol layer]

[The pink phenol layer has denatured protein]

RNA precipitation:

Add 0.5 volume of isopropanol to the supernatant to precipitate the RNA. It is wise to put the tubes in ice for better precipitation.

Centrifuge the tubes at 10,000g for 10 minutes at 4^oC to pellet the RNA.

RNA pellet is transparent. Do not get dishearten, if you do not see the pellet.

Washing:

Carefully decant the supernatant without disturbing the RNA pellet.

Wash the RNA pellet with 75% ethanol.

Dry the pellet at room temperature to evaporate the ethanol

Dissolve:

Dissolve the RNA in DEPC treated Milli Que water.

Take absorbance at 260 and 260 wavelength to estimate the concentration and purity.

In case if 260/280 ratio indicate DNA contamination, treat the RNA with RNAse free DNAse I