Hi Everyone,

I've been trying to isolate RNA from mouse skeletal muscles (soleus, gastro, EDL, etc.) using the QIAGEN RNeasy mini kit. However, I've been having issues with low yield of RNA (10ng/ul in 40 ul). It seems to work well for cardiac muscle, so I'm not sure if it is just because these muscles are small (~10mg). I was hoping you guys could help me out. Here is my current protocol:

1) Mince muscle that was stored in -80 into small pieces, and add into sterile 2 ml tube.

2) Add sterile silicon beads and 600ul RLT (RNeasy lysis buffer) and homogenize for 1 minute in beadbeater ( at 4C).

3) Rest on ice before spinning at 12,000 RPM.

4) Collect supernatant into fresh tube.

5) Mix with cold 60% EtOH.

6) Load onto QIagen column and spin for 30 sec at 12000 rpm. Discard f.t.

7) Add 700ul RW1, spin 30 sec, discard f.t.

8) 2 washes with 500 ul RPE with spin between each.

9) Additional 1 minute spin in fresh tube to dry column,

10) Add 40ul H2O and elute with 1 minute spin.

Thank in advance for your help guys!