Utter precision and remarkable lab techniques are essential when isolating RNA. When compared to fixed and unimpeachable DNA, RNA is easily alterable. As RNA comprises a ribose sugar backbone, it contains exceptionally reactive hydroxyl groups, which ensure that strands are rigorously broken down, made, and reused. Outlying RNA deteriorates quickly and is more prone to contamination by resistant enzymatic ribonucleases, which can be found everywhere.
San Francisco’s California Academy of Sciences curator and chairman in the Department of Mammalogy and Ornithology, Dr. Jack Dumbacher, expressed his views on his work of isolating small RNA and transcriptome to identify metagenomic viral sequences. Contrary to the common belief that views viruses as pathogens, viruses may also possess symbiotic or mutualistic relationships with their hosts. Comprehending these revolutionary relationships in birds is the main aim of Dumbacher’s study.
Dr. Jack Dumbacher stated that generally with virus particles, you are after RNA with low abundance.
Improve RNA Preservation.
Remote areas that are known for their biodiversities such as coral reefs or tropical forests are the areas where the Academy’s collecting expeditions often take place. The major problem which is causing Dumbacher’s team some discomfort is the lack of refrigeration as they are unable to snap-freeze samples with liquid nitrogen. RNA easily deteriorates in Ethanol which is a perfect source for preserving DNA samples. Instead, samples are stored at a 4 M concentration of mixtures containing guanidinium thiocyanate, or they are placed in RNA stabilization reagents. Isolating RNA in samples of liquid can be difficult because of the salt contents if utilizing RNA stabilization reagents.
Ensure that the separation is done perfectly.
Initially, Guanidinium Thiocyanate was used to lyse viral particles and cells in RNA extractions and simultaneously prevent RNase enzymatic activity by altering RNA temporarily. In these cases, some whole viral separations, similar to filtration, must be conducted before lysing to ignore a soup of nucleic acids. Dumbacher’s team collected some samples which are primarily in liquid form, a good separation is important but a little homogenization is also required. The team utilizes 0.2 µ filters and filter tips, similar to the ones found in spin columns in order to filter through smaller virus particles. Intact cells which are comparatively larger are left behind. Other team members work with frozen tissue samples that require homogenization thoroughly and quickly in order to ensure a good recovery. Regardless of this method, some amount of genomic DNA will remain no matter what they do but the amount that gets through is entirely dependent on the user’s technique and experience.
Lower the RNase exposure.
Eliminating interaction between exogenous and endogenous RNase and purified RNA is always a concern in the lab. In the initial stages of the RNA purification process, unrelated sources of RNase are less threatening. However, after the RNA is prepared, the need to protect them by denaturants like the GITC solutions is no more and the RNA is then purified. Furthermore, you want to avoid activating RNases at all costs.
Increase RNA yields.
If you don’t already keep track of your extractions, start doing so without delays. RNA yields vary massively from different samples and tissues. Every sample collected by Dumbacher’s team is generally low in RNA. The amount of RNA gained is entirely dependent on when the bird last ate, what it ate, and whether any chemicals were consumed by the bird.
To enhance the tissue samples with increased RNA yields, avoid unnecessary heat or homogenization. To improve RNA recovery, homogenize in bursts of 30 seconds with 30 seconds rest intervals. When utilizing silica spin filters, surface more water in order to release more RNA from the membrane. Regardless of the lab equipment and technology used in your lab, successful analysis is entirely dependent on good RNA extraction techniques
RNA extraction can be a complicated process. A beginner can face a lot of problems in the process and as mentioned above, precise RNA extraction is entirely dependent on good techniques. With regards to this research blog, if you require any microbiology products, check out MBP Inc’s official website.