Homogenize tissue samples in mortar with excess amount of liquid nitrogen. Measure the weight of homogenized samples.

Transfer 50-100 mg of tissues in 50ml tube having 3 ml of TRIzol reagent (Invitrogen). Homogenize the samples using Tekmars TISSUMIZER or Polytron or equivalent. Always keep the tube in ice so as to prevent the overheating.

Optional Step: Additional step may be required for samples with high content of proteins, fat, polysaccharides or extra cellular materials such as muscles, fat tissue and tuberous parts of the plants. Following homogenisation, remove insoluble materials by centrifugation at 12,000 g for 10 minutes at 2-4 .C. The resulting pellet contains insoluble materials and the supernatant contains RNA. In samples from fat tissue, an excess of fat collects as a top layer, which should be removed. Transfer the clear homogenate solution in a fresh tube.

Phase Separation: Incubate the sample at 15 to 30 .C (RT) for 5 minutes to permit for the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform (high grade) per 1 ml of TRIzol reagent. Shake tubes vigorously for 15 seconds and incubate them at 15 to 30 .C (RT) for 2 to 3 minutes. Centrifuge the samples at 12000 g for 15 mints at 2 to 4.C. Following centrifugation; the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colourless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIzol used for homogenisation.

RNA Precipitation: Transfer the aqueous phase to a fresh tube and save the organic phase if isolation of DNA or protein is desired. Precipitate RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of Isopropyl alcohol for every 1ml of TRIzol used. Incubate samples at 15 to 30 .C for 10 minutes and centrifuge at 12000g for 10 minutes at 2-4.C.

Remove the supernatant carefully without disturbing the pellet. Wash the RNA pellet once with 75% alcohol, adding at least 1ml of 75% ethanol for every 1 ml of TRIzol used. Mix the sample by vortexing slowly and centrifuge at 7500 g for 5 minutes at 2-4.C.

Remove the supernatant carefully without disturbing the pellet. Dry the RNA pellet in air for 10 minutes. Dissolve the RNA pellet in 100 ul of RNase free water. Pipette out few times and transfer in eppendorf tube and store in 70.C.

Total RNA is further cleaned up by using RNeasy mini kit (Qiagen) Cat No:74104

Step 1: 350ul of RLT buffer is required for each total RNA sample. Take the aliquot required in a tube (If you are going to clean 10 samples, calculate for 12 samples and take 12*350= 4.2 ml) and add 10ul of B-Mercaptoethanol (Sigma-Molecular Biology Grade) per 1ml of RLT buffer (42ul for 4.2 ml of RLT buffer).

Step 2: Add 350 of RLT Buffer (B-Mercaptoethanol added) to 100ul of total RNA sample (if the volume is less make up to 100ul) and mix thoroughly. Add 250ul of 100% ethanol. Mix well by pipetting. Do not centrifuge.

Step 3: Apply sample (Should be around 700ul) to an RNeasy mini spin column, sitting in a collection tube. Centrifuge in cold for 20 sec at 8000g. You can apply the flow through in the column for twice to improve the yield. Discard the flow through as well the tube after repeating step 3 twice. Keep the column in a fresh 2ml tube.

Step 4: Apply 500ul of RPE buffer (please make sure that alcohol is added in RPE buffer) on to the column. Centrifuge for 15 sec at 8000g. Discard the flow through.

Step 5: Apply 500ul of RPE buffer and centrifuge for 2 min at max speed. Discard the flow through. Spin again at max speed for a minute. Transfer the column in a fresh RNase free collection tube.

Step 6: Pipette 30ul of RNase free water on to the column. Heat the sample at 55.C for not more than 2 minutes (to get maximum yield). Centrifuge at 8000g for a minute. Repeat step 6 once more to get 60ul of clean total RNA and proceed with QC and OD measurements. It is always to better keep the stock in two-three aliquots (to avoid freeze-thaw cycle) before freezing them in dry ice and transfer them to -80.

All experiments using protocol P-BAIR-1: (E-BAIR-1, E-BAIR-13, E-BAIR-2, E-BAIR-3, E-BAIR-4, E-BAIR-5, E-BAIR-6)