RNA is highly susceptible to degradation by RNAse enzymes. RNAse enzymes are present in cells and tissues, and carried on hands, labware, and dust. They are very stable and difficult to inactivate. For these reasons, it is important to follow best laboratory practices while preparing and handling RNA samples.
- When harvesting total RNA, use a method that quickly disrupts tissue and isolates and stabilizes RNA.
- Wear gloves and use sterile technique at all times.
- Reserve a set of pipettes for RNA work. Use sterile RNAse-free filter pipette tips to prevent cross-contamination.
- Use disposable plasticware that is certified to be RNAse-free. Illumina recommends the use of non-sticky sterile RNAse-free microcentrifuge tubes. These should not be used for other lab work.
- All reagents should be prepared from RNAse-free components, including ultra pure water.
- Store RNA samples by freezing. Keep samples on ice at all times while working with them. Avoid extended pauses in the protocol until the RNA is in the form of double-stranded DNA.
- Use RNAse/DNAse decontamination solution to decontaminate work surfaces and equipment prior to starting this protocol.