Due to the widespread presence of RNases and the intractable properties that are difficult to inactivate. It makes the extraction and purification of RNA and subsequent work very difficult.
What should you know about RNA and RNases?
RNases are very stable and active enzymes that generally do not require the function of cofactors, so RNA is easily degraded in the laboratory.
Do not use any plastic or glass products if the possible RNase contamination is not eliminated in advance.
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?
RNA purification from fibrous tissue
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.
Purification of RNA from FFPE samples
Immobilization and embedding 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
The 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
Purification of RNA from blood samples
Red blood cells in whole blood have no nuclei, and the number of practical cells per milliliter 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.
Purification of free RNA from serum, plasma and other cell-free body fluids
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.
About viral RNA purification
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 a suitable reverse transcriptase.
Dear Customer:
Thank you for your interest in Shanghai Jiapeng's products. In addition to this product introduction, the company also has three kinds of UV analysis, nucleic acid protein detector, gel imaging analysis system, photochemical reactor, constant current pump, automatic partial collector, etc. Introduction, if you are interested in Shanghai Jiapeng products, please contact us. Thank you!
Catering Table,Round Folding Table,Plastic Fold Up Table,Round Plastic Folding Table
ZHEJIANG HUZOLI METAL PRODUCTS CO.,LTD , https://www.zlplasticfurniture.com