DNA/RNA ratio - techniques (Jul/07/2005 )
I' currently working on how to extract RNA and DNA from the same samples into evaluate DNA/RNA ratio.
I extract RNA with TRIzol method and I try to extract DNA from the same sample using the protocol furnished with TRIzol.
Briefly it's an ethanol precipitation of the DNA from the interphase and from the remaining aquaous phase. Then we centrifuge and wash DNA....
My problem is on the first step....ethanol precipitation. Because When we add ethanol, DNA precipitates. But with the centrifugation DNA will pellets but also cells and tissue that remains from the homogeneisation. So how to have only DNA...or maybe I just forget a step (?)
Can someone answer me ?
I have done many RNA and DNA extractions using TriReagent (should be similar to Trizol). The only problem I found for subsequent DNA extraction is low DNA yield. DNA purity seems OK. The following is a modified protocol for DNA back extraction based on TriReagent mannual.
The last centrifugation step is for removing fragments of membranes and other non-DNA stuff.
Back extraction buffer
4M guanidine thiocyanate
50 mM sodium citrate
1 M Tris
To make 40 ml such buffer, add:
18.9 g guanidine thiocyanate
0.588 g sodium citrate
4.84 g Tris
1. after RNA extraction and phase separation, remove any remaining aqueous phase overlying the interphase
2. add 0.5 ml back extraction buffer (for 1 ml TriReagent used)
3. vigorously mix by inversion for 15 sec and store for 10 min at RT
4. centrifuge at 12,000 g for 15 min at 4C
5. transfer the upper aqueous phase to a new tube
6. add 0.4 ml isopropanol
7. mix by inversion and store at RT for 5 min
8. centrifuge at 12,000 g for 5 min at 4-25C
9. remove supernatant
10. wash DNA pellet with 1 ml 75% ethanol
11. centrifuge at 2,000 g for 5 min
12. remove the ethanol and briefly air-dry the DNA pellet (RT for 3-5 min)
13. dissolve the DNA pellet in 8 mM NaOH by slowly passing through a pipette (add an adequate amount of 8 mM NaOH to approach a DNA concentration of 0.2-0.3 ug/ul, typically add 0.3-0.6 ml for 107 cells)
14. centrifuge at 12,000 g for 10 min
15. transfer the resulting supernatant containing DNA to a new tube