RNA Extraction from Agarose

Dominik Haudenschild at TSRI, 2007

1 to 2 discs of fresh sample (6mm x 2mm ~ 65 µL) in 1.5 mL eppendorf tube

Add 620 µL EB1 and homogenize using polystyrene grinder

Use 25 gage needle to completely homogenize sample

Incubate @RT for 5 minutes

Add 400 µL acid Phenol:Chloroform (1:1) and mix rigorously

Centrifuge for 30 minutes µL at 4 o C

Transfer 520 µL of aqueous phase into a new 1.5 mL eppendorf tube containing 520 µL EB2

Mix by inversion and incubate for 10 minutes @RT

Add 420 µL Chloroform and vortex

Centrifuge for 30 minutes µL at 4 o C

Transfer 750 µL of aqueous phase into 1.5 mL eppendorf tube containg 500 µL of cold Isopropanol and mix

Add 420 µL 1.2 M NaCl and mix

Incubate on ice and keep it at 4 o C for 2-3 hours

Centrifuge for 30 minutes µL at 4 o C

Remove all but ~100 µL of supernatant by pipetting or vacuum suction

Centrifuge for 4 minutes @RT then invert tube to remove the rest of the supernatant

Be careful not to discard the clear whitish RNA pellet at bottom of tube

Wash RNA pellet with 1.3 mL of 70% Ethanol

Centrifuge for 4 minutes @RT the remove most of supernatant by pipetting or vacuum suction

Leave ~100 µL of supernatant so that inverting tube could be possible without disturbing the RNA pellet

Centrifuge for 4 minutes @RT then carefully invert tube to remove the rest of the supernatant

Wash RNA pellet with 1.3 mL of 100% Ethanol

This wash is to aid the drying of RNA pellet

Centrifuge for 4 minutes @RT the remove most of supernatant by pipetting or vacuum suction

Leave ~100 µL of supernatant so that inverting tube could be possible without disturbing the RNA pellet

Centrifuge for 4 minutes @RT then carefully invert tube to remove the rest of the supernatant

Air dry RNA pellet or centrifuge with open cap to dry off the remaining ethanol

Dissolve RNA pellet in 102 µL of RNAse free water

Measure RNA concentration using Nanodrop

If cDNA is required

Add 1:10 of 3 M NaOAc (pH 4.7) and mix

Add 2.5 volume of 100% Ethanol and vortex

Incubate on ice and keep it at 4 o C for 2-3 hours

Centrifuge for 30 minutes µL at 4 o C

Remove all but ~100 µL of supernatant by pipetting or vacuum suction

Centrifuge for 4 minutes @RT then invert tube to remove the rest of the supernatant

Be careful not to discard the clear whitish RNA pellet at bottom of tube

Wash RNA pellet with 1.3 mL of 70% Ethanol

Centrifuge for 4 minutes @RT the remove most of supernatant by pipetting or vacuum suction

Leave ~100 µL of supernatant so that inverting tube could be possible without disturbing the RNA pellet

Centrifuge for 4 minutes @RT then carefully invert tube to remove the rest of the supernatant

Wash RNA pellet with 1.3 mL of 100% Ethanol

This wash is to aid the drying of RNA pellet

Centrifuge for 4 minutes @RT the remove most of supernatant by pipetting or vacuum suction

Leave ~100 µL of supernatant so that inverting tube could be possible without disturbing the RNA pellet

Dissolve RNA pellet in 10 µL of RNAse free water

Measure RNA concentration using Nanodrop then make cDNA using SuperScript III

Extraction Buffer 1 (EB1)

100 mM Tris pH 8.0

150 mM LiCl

50 mM EDTA

1.5% SDS

1.5% 2-mercaptoethanol (add prior to use)

Extraction Buffer 2 (EB2)

4M Guanidinium Thiocynate (GITC)

0.75 M Sodium Citrate

10% Lauryl Sarcosine

2 M Sodium Acetate pH 4.0

For 100 mL of EB2 add these in the following order in 35 mL of RNAse free water

47.2 g GITC

22 g Sodium Citrate

8.2 g Sodium Acetate

10.2 mL Glacial Acetic Acid

10 g Lauryl Sarcosine

Equilibrate to 100 mL