Techniques Series: RNA Extraction

June 6, 2013 | Posted by Mamata Thapa in Research |

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RNA extraction involves a series of steps for the isolation of RNA from biological molecules. The final product is used for experiments such as qRT-PCR, microarray, next-generation sequencing, or Northerns.

This technique can aid scientists into addressing a wide range of questions. For instance, what may be the level of mRNA expression of a gene involved in the developmental stage of an invasive insect species? Which steps of rRNA processing are affected in cells depleted of cell cycle proteins? What modifications in tRNAs lead to certain autoimmune diseases? This technique is applicable in a wide range of organismal groups extending from bacteria to humans.

Sample Preparation for RNA Extraction

In order to obtain accurate results in carrying out subsequent experiments, it is critical that you have clean sample of RNA extracted from cells. Proper lab protocol needs to be followed in order to isolate undegraded RNA. This technique is largely problematic in laboratories due to the presence of ribonucleases which are ubiquitous in cells and cause rapid degradation of the nucleic acid.

Before extracting RNA, it is important to extensively sterilize all materials that are in contact with the sample after the cells are harvested. This will requiring autoclaving tubes, water, pipette tips, etc. for a longer period of time than the usual cycle time. DEPC-treated water is also used, which is handy when work with RNA. The diethyl dicarbonate inactivates RNases in water, minimizing the chances of degradation. Clean your bench and pipettes thoroughly with RNase ZAP in order to avoid contamination.

Wear gloves! Even if you carry out the procedure accurately and neatly throughout the extraction, but you touch the RNA sample with a contaminated pipette tip during the last step, this can cause your RNA to degrade. It will be a big headache once you obtain results such as a smear of bands on your Northern blot (indicating degradation) and have to go back to the drawing board, starting from growing fresh cell cultures (keep in mind running RNA gels and Northern blots are additional steps).

Methods for RNA Extraction

The most common method of extracting total RNA is using phenol-chloroform. Once harvested, cells are lysed in different manners, depending on the organism you are working with. For yeast, they have a cell wall and are more difficult to lyse versus gram-negative bacteria cells. Therefore, reagents and materials such as glass beads, phenol, and TESI-SDS are added to a tube and placed in a 95˚C oil bath.

Cells are added to the mixture and tubes are vortexed. Once spun down, the supernatant is transferred to a fresh tube and chloroform is added; tubes are vortexed, and the aqueous phase (top layer containing RNA) is transferred to new tube. One has to be careful not to transfer liquid from the interphase or organic layer (middle and bottom) since these contain DNA and proteins and lipids, respectively. Isopropanol is added and tubes are incubated at -20˚C overnight to allow precipitation of RNA. The following day, tubes are spun, liquid is decanted, and the RNA pellet is washed with 70% ethanol and air dried before being resuspended in water for analysis and storage.

Uses for RNA Extraction

The ultimate goal of this technique is obtain high yields of undegraded RNA for experimental analysis. Since RNA extraction requires several steps of careful handling of samples from the beginning to the end, it can be tedious and laborious. Different companies are now selling kits that cut down RNA extraction time compared to the traditional RNA isolation procedure. Although these products are beneficial, they may not result in high yields of RNA, or the materials may be costly for a laboratory to purchase.

Extraction of RNA is pretty much required for any scientific research these days. With the application of molecular biology technologies, to have the “starting” sample at hand, for example for qRT-PCR or microarray, makes RNA extraction an important technique. Scientists will be able to assess the expression of a gene at a transcriptional level, which will take them one step further if they have studied it at a translational level. It will also allow one to study the various forms of RNAs- mRNAs, rRNAs, and tRNAs and the implications they have in certain human diseases, such as cancer.

There are currently 29 facilities that are listed in our website which provide RNA extraction services. They are listed here: https://www.scienceexchange.com/services/rna-extraction

About the author

Mamata Thapa works at JOVE: Journal of Visualized Experiments. She previously received her PhD in Molecular and Cell Biology from the University of Maryland, Baltimore County, and researched the role of ribosomal proteins in cell cycle progression and its implication in tumorgenesis, and is also a member of the Science Exchange Advocate program.

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