Posted 15 March 2011 - 07:23 AM
Posted 16 March 2011 - 07:32 AM
I would just redo the isolations with 2-4 isolations per sample; pool samples for the DNase treatment (or if you are using columns, pool them into the same column) and proceed.
I haven't worked with tomato seed, but working with pericarp, I found that low RNA yield was due to using too little tissue in the isolation. Most of the tissue weight is due to water and I found that increasing the amount of pericarp increased the yield. For leaves, I normally use ~100mg tissue/1ml of lysis buffer (this is for CTAB based isolation). I increased this to ~200mg/ml for pericap and tripled the yield (using >300mg/ml yielded less than 200mg in some cases).
It's usually a bad idea to increase the amount of tissue, especially for column-based kits, but because pericarp has a high water content you should be able to get away with it. I haven't tried this using a kit like RNeasy, but I'm sure that using ~100-200mg of pericarp wouldn't be a problem (I usually use only 50mg leaf tissue in 0.45ml of RLC buffer with this kit).
Posted 16 March 2011 - 08:35 AM
Posted 16 March 2011 - 09:01 AM