Removal Of Contaminating DNA From RNA Samples
Santosh Patnaik, 1998
RNA isolated from cells or tissue samples is usually contaminated with DNA. Subsequent processing for isolating mRNA usually gets rid of the contaminating DNA.
DNAse I treatment (described here) or acid phenol:chloroform extraction are efficient ways to remove contaminating DNA.
DNA contamination, as revealed by + and - RT RTPCR using standard, control primers (like those for actin or GAPDH) is not important if the control, - RT tube does not yield any PCR product with your gene-specific primers (GSP) while the + one does. Such a scenario may arise if the GSPs span relatively long distances (> 0.5-1 kb) while the control primers are just 2-300 b apart.
It should be remembered that DNAse I supplied by manufacturers may be contaminated with RNAses and proteases.
Many vendors now supply spin-column based DNA removal kits that though a bit expensive are easy to use.
DNase treatment of bulk samples
DNAses from vendors other than Promega may be used as per their suggested protocols.
1. Thaw RNA sample on ice.
2. Adjust RNA concentration to 2 ug/ul by adding DEPC-H2O.
3. Add 0.25 volume 5X First Strand Buffer (used with SuperscriptII RT from Invitrogen). Optionally, add RNAsin or any other RNAse inhibitor (0.25-1 U/ug RNA).
4. Add RQ1 RNase-free DNase (Promega), 2-3 U/100 ug RNA.
5. Incubate at 37 deg for 30 min.
6. Extract RNA with equal volume of phenol:chloroform:isoamylalcohol :: 25:24:1 mixture, vortex for 1 and spin at 12000 g for 2. Transfer aqueous phase to a new tube and repeat.
7. Transfer upper, aqueous phase to a new tube. Precipitate RNA by adding equal volume isopropanol, or sodium acetate to 0.3 M and 2.5 volumes of 100% ethanol. Mix and place at -20 deg for 30 (overnight for better yield).
8. Spin at 12000 g for 5 and wash pellet with 70% ethanol.
9. Air dry the pellet and dissolve in DEPC treated water. Store at -70 deg.
DNase treatment of small samples
1. Use 1U RQ1 DNase per 1-2 ug RNA.
2. Add 2 ul first strand buffer and make final volume to 10 ul with DEPC- H2O.
3. Incubate at room temperature for 15.
4. Add 1 ul 25 mM EDTA.
5. Incubate at 65 deg for 15.
6. Chill on ice. The RNA sample can now be used for reverse transcription.