After insulin and metformin treatment, cells were lysed in lysis buffer (RLT, Qiagen, Rneasy kit) and snap frozen. Subsequently RNA was extracted using RNeasy Qiagen kit as per manufacturer's instructions.

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.

Experiment E-BAIR-7