RNA isolation from mouse heart tissue - (Mar/05/2007 )
I tried isolation of total RNA from mouse heart tissue.
First with Qiagen RNEasy mini kit plus (includes a column to remove genomic DNA), then with the ordinary Qiagen RNEasy mini kit and at last with Mini kit including Proteinase K step (recommended for fibrous tissue).
Photometer and RNA gel say that there's no RNA in my isolate. The only peak I get is at 220/230nm.
Do I still have too much protein in my prep so my column is full of protein and no RNA get's eluted?
(Other possibility of course: all the RNA is degraded in the sample)
In general for RNA extraction with column:
1. check that you use RNA (DNase and Rnase free) only materials and reagents (keep all apart).
2.weight the sample if possible (over ice) and use the correct kit for the quantity, some times we understimated and use the wrong column.
3.Be 100% sure that all is homogenized (no sign of tissue visible!!) or the column could get clog.
4. when elute add first half of the buffer or water, let stand for few min, centrifuge, repeat.
hope this help
Try switching to trizol. 250mg tissue to 5ml trizol. Use a polytron to grind up the muscle.
That should do it.