RNA extraction from hairy roots of common bean (Phaseolus vulgaris L.) and cDNA synthesis

Macerate root tissue using liquid nitrogen.

Load 100mg of macerated tissue into a 1.5 mL Eppendorf tube and add 1mL of .

Mix by vortexing0h 0m 15s and incubate for 0h 5m 0s at room temperature.

Add200µL, mix by vortexing 0h 0m 15s and incubate for 0h 3m 0s at room temperature.

Centrifuge 11800rpm,4°C

Transfer the aqueous phase to a new 1.5 mL Eppendorf tube.

Add 500µL of isopropanol, mix by immersion, and incubate 0h 10m 0s -20°C .

Centrifuge 11800rpm,4°C

Transfer the aqueous phase to a new 1.5 mL Eppendorf tube.

Add 500µL of 4 M LiCl and rise the pellet, do not resuspend, vortex slowly.

Centrifuge 5900rpm,4°C,0h 0m 0s``0h 20m 0s

Discard the LiCl phase.

Add 500µL of tris-EDTA buffer 8 . Resuspend RNA by vortexing.

Add 500µL and mix by vortexing.

Centrifuge 5900rpm,4°C

Transfer the aqueous phase to a new 1.5 mL Eppendorf tube.

Add 500µL of isopropanol and 66µL of 3 M sodium acetate 5.2 Mix by immersion and incubate -20°C

Centrifuge 11800rpm,4°C

Discard the aqueous phase and vacuum or air dry the RNA pellet.

Resuspend RNA pellets using nuclease-free water. Preferably, use DEPC-treated water.

Check the integrity of RNA in a 1% agarose gel treated with bleach.

Prepare a dilution (1/10) of each RNA sample and quantify the concentration using a or an equivalent instrument.

Prepare one aliquot 10µLof each RNA sample at 10ng/μl

Add 1µL of

and 1µL of the corresponding buffer (10X) to each RNA sample.

Incubate samples0h 30m 0s 37°C

Add 1µL of 10millimolar (mM) to each sample and incubate0h 5m 0s 70°C, then immediately incubate 70On ice .

Prepare a mix containing 1µL of, 4µL of the corresponding buffer (5X), and 2µL . Add 7µL of the mix to each RNA sample.

Incubate 1h 30m 0s at42°C and 0h 10m 0s 70°C .

Store cDNA samples at -20°C .