I am trying to Isolate RNA from sugarcane. The objective is to study the involvement of genes in red rot diseases.

Plant stems are inoculated with Red Rot fungus inoculum. Plants are mature and it's been 2 years of their growth.

The protocol I follow is as

1.                  Add 0.5 ml of cold (4ºC) Plant RNA Reagent (TRIzol from Invitrogen) for up to 0.1 gm of frozen, ground tissue. Resuspend the sample thoroughly by briefly vortexing or flicking the bottom of the tube.

2.                  Incubate the tube for 5 min at room temperature. Lay the tube down horizontally to maximize surface area during RNA extraction.

3.                  Centrifuge for 2 min at 12,000 x g, transfer the supernatant to an RNase- free tube.

4.                  Add 0.1 ml of 5 M NaCl to the clarified extract and tap the tube to mix.

5.                  Add 0.3 ml of chloroform. Mix thoroughly by inversion.

6.                  Centrifuge the sample at 4ºC for 10 min at 12,000 x g to separate phases. Transfer the top, aqueous phase to an RNase-free tube.

7.                  Add to the aqueous phase an equal volume of isopropyl alcohol. Mix and let stand at room temperature for 10 minutes.

8.                  Centrifuge the sample at 4 ºC for 10 min at 12,000 x g.

9.                   Decant the supernatant, taking care not to lose the pellet, and add 1 ml of 75% ethanol to the pellet.   Note: The pellet may be difficult to see.

10.              Centrifuge at room temperature for 1 min at 12,000 x g. Decant liquid carefully, taking care not to lose the pellet. Briefly centrifuge to collect the residual liquid and remove it with a pipette.

Add 10-30µl RNase–free water to dissolve the RNA. Pipette the water up and down over the pellet to dissolve RNA.  Store at -70 ºC.

Before starting the protocol we need pestle, mortars, tips both 1ml and 200ul, Eppendorf, and PCR tubes washed with DEPC-treated water. DEPC 1ml added to 1-liter water makes the DEPC treated water.

5ul of loading dye and 5ul of RNA sample was run on 1% Agarose gel in TAE buffer for 50 min at 60 voltage, 1kb ladder was used and I did not use the Denaturation method for gel.

Attached are the pictures and I am not getting any results kindly help me with how to proceed.