DNAse Treatment Of Total RNA

Overview

Most RNA extractions procedures yield RNA, with minimal or no contamination with DNA, that is suitable for applications such as northern blot, constructions of cDNA libraries or microarray hybridizations. However, applications that rely on specific amplification of cDNAs by PCR to monitor or quantify gene expression require RNA preparations devoid of any DNA contamination. For these applications digestion of contaminating DNA is recommended. This protocol makes use of Quiagen kits to hydrolyze DNA, using an RNAse free DNAseI, and purify the cleaned RNA free of DNA and free of DNAseI activity.

Procedure

1. Transfer the total RNA to a clean 1.5 ml tube and adjust the volume of the total RNA sample to 100 ul with RNAse free water.
2. Add 350 ul of RLT buffer and mix by pipetting.
3. Add 250 ul of 100% ethanol and mix by pipetting.
4. Apply the sample to an RNeasy column and centrifuge for 30 seconds at 10,000 rpm.
5. Discard flow-through and apply 350 ul of RW1 buffer to the column and centrifuge for 30 seconds at 10,000 rpm.
6. Discard flow through and place column back in the collection tube.
7. For each sample to be treated, prepare a DNAse solution by diluting 10 ul of DNAseI (from the kit) into 70 ul of RDD buffer (from the kit) mix by pipetting (do not vortex).
8. Deposit the DNAseI solution directly onto the center of the membrane without touching the membrane with the pipet tip.
9. Incubate for 15 minutes at room temperature.
10. Apply 350 ul of RW1 buffer to the column and centrifuge for 30 seconds at 10,000 rpm.
11. Discard flow through and place column back in collection tube.
12. Apply 500 ul of 80% ethanol (prepared with RNAse free water) and centrifuge for 30 seconds at 10,000 rpm.
13. Discard flow-through and apply another 500 ul of 80% ethanol (with RNAse free water) to the column and centrifuge for 30 seconds at 10,000 rpm.
14. Discard flow through and place column back in the collection tube and centrifuge for 1 minute dry the membrane.
15. Transfer the column into a new 1.5 ml collection tube and pipet 50 ul of RNAse-free water directly onto the center of the membrane without touching the membrane with the pipet tip.
16. Centrifuge for 1 minute at 10,000 rpm to elute RNA
17. Measure concentration of cleaned RNA solution by measuring OD260 of a 1/100 dilution with a spectrophotometer and run 1-2 ug of RNA on a 1.3 % agarose/formaldehyde gel to check integrity of RNA.

Reagents/Solutions