Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Gene expression profiling using RNA extracted from healthy versus diseased tissues is a powerful tool in the investigation and identification of disease biomarkers. Increasingly, researchers are looking for suitable tissue samples to study biomarkers for cancer, for example. Fresh tissue, such as from biopsies, is ideal scientifically but not logistically, as biopsies are not a dependably steady source, and preservation conditions and times from actual biopsy to RNA extraction may vary. This quandary has renewed interest in extracting RNA from formalin-fixed, paraffin-embedded (FFPE) samples, taking advantage of the many well-documented samples from patients in disease studies and clinical trials.
Such treasure troves of tissue samples are a boon to biomarker researchers. Not only have they already been collected, preserved, and organized, but they can be stored for years until needed. Enter the innovative methods to extract useful RNA from these archived samples. Even though the fixation process itself degrades RNA to some degree, resulting in smaller RNA fragments compared to fresh tissue samples, the benefits of using FFPE samples often outweigh this downside. You can still extract and purify the RNA and use it for expression profiling with quantitative (a.k.a. real-time) reverse transcription PCR (qRT-PCR). Kits available to streamline this process make it easier, with automation-friendly formats that allow for increased throughput.
Why is it hard to extract FFPE RNA?
When the original fresh sample is fixed with formalin, many chemical events occur that are not easily reversed when you want to use the sample later. For example, the DNA, RNA and protein molecules become cross-linked, which increases their rigidity and makes them more vulnerable to mechanical shearing forces. Extracting RNA molecules involves freeing them from this cross-linked mass. Some extraction assays can take as little as 30 minutes, depending on the protocol. However, the proteinase K digestion step (used to digest away proteins from nucleic acids) may be more effective if left overnight. [1] It is also more effective at a higher temperature, such as 60°C. Generally, there are two main types of protocols—separation by spin filters or magnetic beads.
Spin (centrifugation) filters
In the spin filter method, RNA extraction begins with paraffin removal. Some kits do this by washing with xylene and ethanol, or phenol andchloroform—or with a less toxic “lysis buffer” formulated by the manufacturer. Following paraffin removal is a proteinase K digestion step to digest away proteins from the nucleic acids. The spin filter method then uses small filter columns within centrifuge tubes that filter the sample as it is driven through the column during centrifugation. The DNA is removed by the filter, and the eluted RNA is at the bottom of the tube. Often it’s particularly important to remove genomic DNA from the RNA sample, so many kits also include a DNase incubation before filtering to remove virtually all the genomic DNA from the RNA.
Kits that use this general method can be fast and convenient, and in some cases that may be sufficient. However, if you obtain a lower RNA yield than expected, lengthening your protocol by incubating with proteinase K for longer and/or at a higher temperature often improves yield.
Magnetic beads
The magnetic bead method is especially amenable to automated, higher-throughput protocols, and it sometimes provides larger yields. With this method, particles such as magnetized beads are coated with polymers that have a high affinity for nucleic acids. After sample digestion, the coated magnetic beads are incubated with the nucleic acid sample. A magnetic field applied to one side of the sample tube isolates the beads (with nucleic acids attached), while the remaining sample material is removed. A subsequent DNase incubation removes DNA sticking to the beads, leaving an RNA sample behind.
In a study of RNA extraction that compared magnetic bead-based to spin column protocols, Ribeiro-Silva and colleagues found that both methods gave similar RNA quality, but the magnetic beads were faster and yielded more RNA. [2] Another possible benefit of magnetic bead separation is that, compared with centrifugation, it does not create mechanical shearing forces that could potentially degrade nucleic acids. (Spin methods are widely used, however, so this additional shearing force may not necessarily be a problem.)
RNA and DNA from one sample
If you want to extract both RNA and DNA in separate fractions, check out kits that use a twist on the spin column method. After removing paraffin and digesting away proteins, a centrifugation step concentrates the RNA into the supernatant and the DNA into the pellet. Then you separate these fractions into separate sample tubes, treat them to enrich either DNA or RNA and then spin them through the filter columns separately. This results in purified, eluted RNA and DNA samples.
Working with RNA is always challenging, but today’s tools minimize discouragement. As Ribeiro-Silva and colleagues suggest, if one method doesn’t yield the expected quality or quantity of RNA, try another kit or another protocol altogether before throwing in the towel.
References
1. Abramovitz M, et al. Optimization of RNA extraction from FFPE tissues for expression profiling in the DASL assay. Biotechniques 44: 417-423. (2008)
2. Ribeiro-Silva A, et al. RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies. BMC Mol Biol 8: 118. (2007)
The image at the top of the page is from Life Technologies' PureLink™ FFPE RNA Isolation Kit.