RNA extraction - (Mar/15/2011 )
I have extracted RNA from tomato using seed and pericarp. For the seed, I got the RNA concentration around 45-55ng/ul (in 40ul) and for pericarp I got 27-35ng/ul (in 40ul). Are these concentration low or high?? I have ran the gel to look at my RNA and the bands are very clear and quite thick. And then I have to do the DNase free treatment which can decrease more of my RNA concentration and then proceed to the cDNA synthesis where I have to use around 1ug of RNA . I'm not sure whether I have enough RNA to synthesize my cDNA. Any suggestion on how to increase my RNA concentration and how can I synthesize my cDNA using these samples...
If you must have 1ug for cDNA, you probably won't have enough with these samples after treatment with DNase and cleanup. If you did multiple isolations from the same sample, you could pool them and that would give you enough.
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).
thanks for your suggestion before. Before this, I extracted RNA using qiagen kit with 0.1g of sample. I'll try increase my sample and see what happen
Generally, it is better to use only 0.05g (50mg) of fresh leaf tissue with the Qiagen kit; you may get better yield from seeds if you go down to 20-50mg.