Preparing RNA has traditionally been a tedious, toxic, and nail-biting endeavor involving phenol/chloroform phase separation and alcohol precipitation. These days there are many much easier ways to get RNA ready for downstream applications such as sequencing and reverse transcription—using techniques that are quickly mastered by even laboratory novices. And while organic extraction is still considered to be the gold standard, different kits and adaptations to the protocol has made even that process far more routine and less daunting than it was.
We look here at kits, reagents, and procedures the modern lab uses to efficiently go from tissue to application-ready RNA.
Preliminaries
RNA is labile, and it’s important that it remains as intact as possible starting at the very beginning of the preparation process. If the tissue is not going to be used immediately upon harvest, make sure to take steps to preserve its integrity. The preferred method is flash freezing in liquid nitrogen, points out Janette Lamb, assistant director of the University of Pittsburgh Genomics Research Core. If that’s not an option, put the sample in a preservative such as RNAlater “and use a small enough piece —if it’s too big a piece, it won’t preserve properly.”
The process of releasing the RNA from the tissues may require different procedures. Some, like blood or cell lines, can simply be lysed, while others may need to be minced with a scalpel, sonicated, or homogenized by grinding with glass beads, for example, to provide mechanical disruption.
Most isolation—whether using kits or homebrew methods —takes place in the presence of denaturants such as chaotropic salts, detergents, or phenol that will inhibit nucleases.
RNA purification generally follows one of several general methods (or sometimes a hybrid or combination of methods). Most common is organic extraction, in which the hydrophilic RNA is separated from other components of the sample such as DNA and protein. Spin columns and magnetic beads can be used to capture RNA onto a solid surface. And, while not strictly “purification,” direct lysis (often into a DNAse-containing buffer) can be used directly for some downstream applications that can tolerate a cruder input, or it can be further purified.
TRIzol
Guanidinium thiocyanate-phenol reagents and kits—formulations of which are sold under variety of trade names, but commonly referred to as TRIzol™—have largely become the organic extractant of choice for preparing RNA. TRIzol can be added directly to tissue or cells. Once the cells are homogenized, chloroform is added, and the solution is mixed and centrifuged to facilitate phase separation. The upper, aqueous, phase containing the RNA can be pipetted off into a clean tube, leaving behind the DNA (at the interface) and proteins (in the organic phase).
“If you’re using TRIzol you pretty much only get RNA,” says Lamb, who relies on the reagent for standard extractions. “And it’s cheap,” she adds.
“It works great … but if you have a lot of samples to prep it’s a real pain,” opines Carol Kreader, R&D fellow at MilliporeSigma. “It’s a phenol-chloroform extraction, and you have to get rid of the hazardous waste.”
Zymo Research’s Direct-zol™ RNA MiniPrep Kit allows samples in TRIzol to be processed by spin column. “It’s much faster than what you do with phase separation, and gives similar results,” notes Ryan Sasada, a research associate at Zymo.
Spin columns
William Fulton, senior lab manager in pediatric surgery at Johns Hopkins School of Medicine, uses TRIzol to get RNA out of 3D organoid culture “because it helps to dissolve it all into solution better.”
Otherwise, his lab primarily uses silica-based RNEasy spin column kits from Qiagen. Under the appropriate buffer conditions “bind-wash-elute” silica (or glass fiber, or ion-exchange) membranes will selectively bind RNA. “We use the kit because it just makes it easier for various scientists in the lab—TRIzol is pretty nasty stuff,” he says. There are lots of comparable kits out there nowadays, but they stick with the RNEasy to allow for historical comparisons, explains Fulton. Using a repeater pipette, throughput is mainly limited by the centrifuge capacity.
Biocompare’s Transfection Search Tool
Find, compare and review transfection
tools from different suppliers Search
Spin columns can become clogged with particulate matter from the sample, and have only a limited binding capacity. Large nucleic acids such as genomic DNA may be retained along with the RNA, so some researchers will treat the sample with DNAse either on-column or post-purification.
Lexogen’s SPLIT RNA Extraction kit performs a phenol-chloroform extraction using a phase locked gel to help keep the phases separate. The resultant aqueous phase is then further purified on a silica column. Adjusting the amount of alcohol will bias the recovery toward long or total RNA.
Magnetic beads
Due to their solution-based kinetics, magnetic particles are very efficient at capturing RNA. A wide variety of surface chemistries are available to target mRNAs, for example, or a panel of specific RNAs. The RNA can be eluted in a small volume, and reverse transcription can be done right on the beads using the oligo-(dT) capture probe as the primer. “That’s a nice way to do it if you have a lot to do, especially, and if you’re working with low levels of starting material, Kreader points out.
“The nice thing about beads is that they’re scalable,” she adds, “but they’re generally more expensive.”
Lysis
Tissues and cells can be processed by directly lysing them in buffer designed to disrupt the cell membrane and stabilize the lysate. Because binding and elution is not part of the process, recovered samples may avoid some biases inherent in other methods. But because the output is not purified, concentration cannot be directly assessed spectrophotometrically.
Arcis Biotechnology recently introduced a two-tube, three-minute sample prep kit that uses detergents to put pores into lipid bilayers, releasing nucleotides into the lysis buffer. “All the nucleic acids aggregate together in a clump … and are stabilized so they can’t be attacked by nucleases,” explains Andrew Birney, global life sciences product manager for Cole-Parmer, which distributes the kit. RNA will remain stable for six days at ambient temperature. “Then when you take an aliquot of the lysis buffer and put it in the wash buffer, all the nucleic acids string out and become linear.” The kit yields total nucleic acid; it does not discriminate between DNA and RNA.
Miscellany
Many protocols and kits will yield the entire spectrum of RNA, from diminutive microRNAs to multi-kilobase-long mRNAs. But be careful. “There are a lot of [silica-based] kits historically called ‘total RNA’ that you lose most of what’s less than 200 bases long,” cautions Kreader. She recommends TRIzol- or silicon carbide-based kits for small RNAs.
Check the input requirements. While most protocols and kits are designed for thousands or millions of cells, several kits are now being marketed as being capable of purifying RNA from single cells.
For the most part RNA from the above methods will be good enough and pure enough for next-generation sequencing without having to get rid of any DNA contamination. But for reverse transcription (or when sequencing low abundance RNAs), DNAse treatment is recommended. And for added piece of mind, it’s best to design primers that span a splice junction.
Lamb’s parting words: “If you use a kit in the lab and it works, don’t make any changes.”