Quantitative PCR (qPCR) enables precise, accurate DNA quantification. The quality of the starting material is of paramount importance to obtaining reliable, consistent qPCR data. Here, we provide sample preparation considerations to help obtain accurate and consistent results when performing 5’ nuclease assays.
RNases and DNases can quickly degrade samples as well as oligonucleotide primers and probes. They are ubiquitous and can be difficult to eliminate. Therefore, precautions must be taken to ensure that samples are protected from these nucleases.
- Thoroughly clean sample preparation areas with 10% bleach solution before you start. Bake glassware and treat non-RNase-free plastics with a 0.1% solution of DEPC for 2 hours followed by rinsing with RNase-free water. Alternatively, IDT provides Nuclease Decontamination Solution, which irreversibly inactivates nucleases and can be applied to most lab surfaces.
- Always wash your hands and wear gloves before handling containers used for RNA sample preparation and storage.
- Use nuclease detection reagents such as RNaseAlert® and DNaseAlert™ Kits to test your samples and reagents
- If you find contamination in your samples, replace all reagents and stock buffers and thoroughly clean sample preparation areas.
- You can add RNase inhibitors to block the action of some ribonucleases; DNase can be inactivated by heat treatment.
- Regularly decontaminate equipment, such as pipettors and vortex mixers.
Treat your RNA sample with DNase to prevent amplification of genomic DNA.
Be sure to use the isolation method appropriate for your sample type.
- Use the same isolation and quantification methods for all samples being assessed in the same experiment.
- For maximum recovery, use organic extraction methods such as TRIzol® (Invitrogen) or QIAzol® (Qiagen) for small RNAs and miRNAs.
- For high-throughput processing, use solid phase kits.
- Use reagents that are never in contact with potentially contaminating DNA and nucleases by keeping enzyme mixes, nuclease-free water, primers, probes, pipettes, tubes, filter tips, and PCR plates away from areas in which template is present.
For short-term storage (up to a few weeks), store RNA resuspended in water or buffer at –20°C. TE buffer (10 mM Tris, 0.1 mM EDTA) is excellent for this purpose. For longer term storage, precipitate the RNA (1/10 volume 3 M sodium acetate; 2 volumes 100% ethanol) and store it in ethanol at –20°C or –80°C. Before use, remember to pellet and resuspend any RNA that has been stored as a precipitate.