RNA extraction and purification considerations

Due to the widespread presence of RNase and the intractable properties that are difficult to inactivate. It makes the extraction and purification of RNA and subsequent work very difficult.

1. RNases are very stable and active enzymes that generally do not require the function of cofactors, so RNA is easily degraded in the laboratory.
2. Do not use any plastic or glass products if the possible RNase contamination is not eliminated in advance.
3. Purified RNA is dissolved in RNases-free water and stored at –20°C or –70°C for up to one year without RNA degradation

For RNA purification using the RNeasy Kit, no additional DNase digestion is required, as QIAGEN's patented RNeasy silica membrane technology removes most of the DNA efficiently. However, for some RNA-based sensitive applications, we recommend using the RNase-free DNase Set (see the RNeasy product specification for digestion) to ensure that the resulting RNA is free of any DNA contamination.

The main problem with RNA work is to prevent RNase contamination. In addition to the need to pay attention to RNase during operation, what are the points for attention when performing RNA purification for different types of samples?

Isolation of RNA (such as heart or muscle tissue) from fiber-rich tissues is difficult because the contractile proteins, connective tissue, and collagen in the fibrous tissue interfere with the RNA purification process. Therefore, in order to remove the effects of these proteins, tissue samples require the use of proteases or phenol-containing lysates. At the same time, the above treatment conditions can not cause RNA degradation, QIAGEN recommends using RNase-free proteinase K for digestion.

Immobilization and entrapment processes can result in severe degradation and chemical modification of nucleic acids, and the nucleic acids we purify from FFPE samples typically have a lower molecular weight than fresh or frozen tissue. The extent of nucleic acid degradation/fragmentation depends on the type of sample and age, and is also strongly dependent on fixation, embedding and storage conditions.

Please follow the recommendations below to treat FFPE samples to reduce RNA damage.

• Remove tissue samples and fix them as soon as possible
• The thickness of the tissue sample should not exceed 5 mm, and the fixed time should not exceed 24 hours.
• Paraffin embedding with high quality reagents, do not use additives
• Avoid staining the sample if possible
• Conditions for storing FFPE samples should be appropriate, such as storage at 4 degrees for up to 1 year, still purified to high quality RNA
• Dewaxing with a suitable dewaxing solution
• There is a step of reverse cross-linking during RNA purification to maximize the release of RNA molecules.
• Remove genomic DNA contamination

The red blood cells in the whole blood have no nuclei, the number of practical cells per ml of blood is small, and the nucleic acid yield is relatively low, so the target of separation from whole blood is white blood cells. In addition, contaminants such as the anticoagulant heparin and EDTA and naturally occurring enzyme inhibitors must be removed, all of which interfere with downstream RNA analysis. QIAamp RNA Blood Kits are highly suitable for purifying high quality RNA from human blood samples, eliminating RNases, contaminants and enzyme inhibitors while maximizing genomic DNA contamination.

RNA, especially miRNA, can be present in serum, plasma, urine, and other body fluids, and is also present in the supernatant of cell culture fluids. The extracellular RNA content is much lower than intracellular RNA, but it is relatively stable and has a half-life of about 2 days in human whole blood. However, repeated freeze-thaw cycles still result in poor purification. Carrier RNA is recommended for purification to improve the efficiency of free RNA recovery.

Some viruses have single- or double-stranded RNA genomes. Viral RNA is usually isolated from acellular body fluids, and the amount of viral nucleic acids in these samples is very low. Viral particles may need to be concentrated by ultracentrifugation, ultrafiltration or precipitation. It is recommended to use carrier RNA during purification to improve the efficiency of recovery of viral RNA. It is also recommended to reverse transcribe certain viral nucleic acids containing complex secondary structures using suitable reverse transcriptases.

The above information is from the official website of QIAGEN.