RNA extraction requires diligence. A little carelessness can destroy the specimen or contaminate the sample. RNA extraction has been a challenge that many labs have to deal with. In the process, the RNA has to be carefully extracted from the DNA. Each sample brings with it different challenges.
For instance, several microbes are immune to the usual chemicals used in the process. Also to enzymatic Lysis. Other samples such as feces or plant-related specimen can contain substances which can inhibit the process by co-precipitating by the RNA. This can cause problems for the commonly used RNA extraction method- Quantitative reverse transcription or RT-qPCR.
However, the most common problems that the labs encounter for RNA extraction are:
This is amongst the most frequent problems for RNA extraction. This primarily happens for two reasons. Firstly, RNA comprises a weaker structure primarily because the main components of RNA are ribose units. Inherently weaker and more susceptible to change structures compared to DNA. This makes it difficult to capture RNA in the desired form.
Secondly, enzymes in RNA (RNases) are extremely difficult to remove. (Jonathan Houseley, 2009)There are processes to tackle the problem for instance, with autoclaving, a bacterial solution can annihilate the bacteria, yet, it can leave traces of RNases. The problem is that even minute traces, which might seem negligible, can degrade the RNA.
Other common problems include:
Low purity of RNA
To ensure that you manage to collect the best RNA samples via RNA isolation, here are the best practices from industry veterans. Down below, we shall see the best practices to extract the best possible yield of RNA while avoiding the above-mentioned problems:
As mentioned above, RNA structures are weaker compared to DNA structures due to the ribose units. Moreover, RNA samples usually have a high percentage of RNases, which can significantly degrade the RNA. The solution to the problem lies at the moment of sample collection. When collecting the sample, the best practice is to stabilize the sample the moment it is collected.
Traditional methods include putting the sample in a freezer with a -80 degree temperature. Liquid nitrogen is another alternative that’s commonly used at labs to preserve the sample. However, these methods aren’t always viable as they put risks to damaging the nucleic acids present in the RNA sample. Also, researchers do not usually have immediate availability to the above-mentioned resources.
Recommended methods by industry experts are:
- The best approach is to thwart the RNases completely via prompt solubilization. This should be done via lysis buffer and immediately followed by freezing the sample.
- Immerse the sample in a stabilization agent. This way, you can protect vital components in the RNA for considerably longer periods. This method is efficient for those who don’t have immediate access to freezing facilities.
Samples are the best if Lysis is complete
To extract the best quality of RNA and to have its maximize yield, the preferred method should be complete Lysis of the sample. The challenge here arises that not all samples respond the same way to Lysis. To ensure maximum efficiency, a careful fusion of lysis buffer and mechanical Lysis in the right amount will bring the process to fruition. Enzymatic Lysis is another option that technicians can have up their sleeves to have the best RNA yield.
Eliminating DNA contamination
DNA can easily contaminate RNA samples. Visualizing RNA samples for possible traces of DNA is the quickest and the most efficient way to acquire the best samples that are not contaminated by DNA. Using the right apparatus such as premium Deep Well Plates and RNA extraction kits is helpful to get the job done.
Substandard products can reduce the sample quality as well. Using premium kits is one sure way to eliminate and avoid DNA contamination.
RNA Isolation is a tricky process and should always be dealt with extreme caution. Use the best advice by the industry experts to master the art of RNA Isolation.
Jonathan Houseley, a. D. (2009). The Many Pathways of RNA Degradation. 136(4), 763 – 776.