Hi! I try to make EMSA to study RNA/protein complex. The RNA length is 427 bp and it is GC rich.

I made RNA from in vitro transciption and DNAse treatment (Promega kit) and purifyed it with ZymoResearch RNA clean & Concentrator-5. After purification I measured the concentration with NanoDrop: C = 1998.3 ng/uL, 260/280 = 2.11 , 260/230 = 2.32 and Qubit: C = 2840 ng/uL.

I also made 2% agarose gel with TAE and load 5 uL of RNA before cleaning (on the left) and ~284 ng of RNA after cleaning (on the right). I see 2 bands after cleaning, but I an not sure about the size of my RNA because I used DNA markers and non-denaturating gel. So, I decided to continue with EMSA.

For EMSA I used 6.5% actylamide gel with 25 mM Tris-HCl (pH=8.8) supplemented with 192 mM glycine. Runnig buffer is 25 mM Tris-HCl (pH=8.8) with 192 mM glycine. I pre-run the gel for 1.5 h, 100V, constant voltage.

I prepared reaction buffer (2.2 uL DEPC water, 2 uL 20 mg/ml BSA, 0.2 uL 0.1M DTT, 0.6 uL 0.1M ATP, 1 uL RNAs and riboneclease inhibitor (Promega), 4 uL Binding buffer (hand made). I incubated my probes 30 min +37oC in thermocycler and then add 2x RNA loading buffer (hand made) and run electrophoresis on ice 2 h, 100V, constant voltage.

I stained gels with SybreGold for 30 min in the running buffer.

1st image - RNA free 500 ng/lane, RNA/0.5 uL protein, RNA/1 uL protein, RNA/2 uL protein, RNA free 1000 ng/lane.

2nd image - 2% agarose gel with RNA: 1-DNA markers, 2-RNA before cleaning, 3 - RNA after purification.

Why I see multiple bands in EMSA? Does my RNA ok or it is degraded during EMSA?