Analysis of RNA provides the gateway to deciphering cells’ intricate workings. Such information is critical for dissecting gene expression, biomarker analysis, and responses to therapeutics. Dealing with RNA, however, can be tricky. Degradation, limited amounts of sample, and newly emerging RNA subsets represent significant challenges.
New or improved technologies for RNA sample preparation are targeting more efficient processing of limited amounts or degraded RNA as well as simultaneous stabilization and purification of RNA, DNA, and protein. Additionally, experts suggest that the noncoding transcriptome is emerging as a new frontier in biology and a major player in drug discovery.
Profiling an RNA sample will produce results only as good as the RNA employed. It’s another example of the principle junk in, junk out. “Sample quality is often the largest variable that affects the quality of transcript-profiling data and the least appreciated,” explains Eric R. Fedyk, Ph.D., associate director, drug safety evaluation, Millennium Pharmaceuticals.
“The first issue to address is biological quality. That means is the sample representative of what I want to examine. For example, what percent of the biopsy is composed of tumor? You answer this by characterizing the samples via histology and confirming that the serial sections used for transcript profiling contain signatures that are characteristic of the phenomenon of interest; that is levels of oncogene.
“The second consideration is transcript quality,” Dr. Fedyk continues. “Transcript quality is often better characterized than biological quality. It is therefore imperative for investigators to understand these types of variables, so that they can develop an optimal experimental design for yielding the highest quality of data possible.”
Other issues are related to deciding how to collect and store samples. “Flash frozen is the preferred medium for storage,” says Dr. Fedyk. “However, the needs of the transcript profiler are often secondary to the needs of the pathologist, who prefers formalin-fixed, paraffin-embedded (FFPE) samples, because this medium preserves tissue morphology much better than frozen.”
Targeted Sample Preparation
Regardless of the method to isolate samples, Dr. Fedyk stresses that, “It’s important to remember that kits have an inherent bias in what type of RNA they isolate based on their technology. This is readily observed via clustering analysis of microarray datasets from RNA reference standards. Therefore, one should evaluate several kits before beginning a project and then stick with a single kit for the duration.”
Matching the right RNA sample purification method to the downstream application may spell the difference between success and failure, according to Todd Peterson, Ph.D., vp of cloning and protein expression R&D at Invitrogen. “Many scientists know how to isolate total RNA, but these methods may not be sensitive enough for certain applications such as microRNA analysis. Also, if you are studying preserved samples such as formalin-fixed, paraffin-embedded tissues, you must deal with chemical crosslinking reagents and sample degradation.”
Invitrogen offers a number of enrichment and purification approaches to help with these special issues. For example, their RiboMinus kit employs sequence-specific subtraction of ribosomal RNA (rRNA) from total RNA. “A total RNA preparation may have as much as 95% of the mass as rRNA,” says Dr. Peterson. “For many applications, this isn’t a problem but when you need higher sensitivity, such as when using microarrays, it is a big problem.”
Many applications center on miRNA, but many kits have poor recovery when using spin columns, according to Dr. Peterson. “Our PureLink miRNA isolation kit was developed for isolation of this important subclass within the total transciptome.
“Isolating good-quality RNA from FFPE specimens can be particularly daunting,” says Dr. Peterson. “There is a vast universe of tissue types, fixation protocols, and agents that impact the extent of crosslinking across RNA targets with naturally different half-lives. Our PureLink FFPE RNA isolation kit provides a high degree of purification and recovery in part because of an important, optimized step where the sample is heated to reverse the chemical crosslinks in FFPE samples.”
Even with these developments, Invitrogen is continue to work on technologies to keep up with the fast growing RNA isolation market is important. The company has identified RNA analysis in the noncoding transcriptome as a key area, according to Dr. Peterson. “There is a large mass fraction of total RNA transcribed that represents noncoding sequences in the genome. This massive population of noncoding RNA is cell- and tissue-specific, regulated, and appears to have structural and regulatory functions.”
Big Results from Tiny Samples
Often the most valuable samples collected in clinical and discovery work have the least amount or poorest quality of RNA. The challenge for working with such samples is the ability to assess the miniscule amount of RNA present.
NuGEN Technologies developed the WT-Ovation™ RNA amplification systems powered by their Ribo-SPIA® technology for amplifying the whole transcriptome from small amounts of sample, even if it’s degraded.
The ability to perform large-scale gene-expression studies from picograms of material is revolutionary and opens up new doors, points out Gianfranco de Feo, Ph.D., senior director, strategic marketing. “Usually, micrograms are needed for such work. However, our technology overcomes these sensitivity issues and provides rapid, high-fidelity RNA amplification for gene-expression analysis regardless of if the starting RNA is degraded or present in very low amounts.
The process includes three steps. Initially total RNA is used to prepare single-strand cDNA with a chimeric DNA/RNA primers and reverse transcriptase that allows the entire transcriptome to be represented. Next, the mRNA is fragmented in the cDNA/mRNA complex, and DNA polymerase generates a second-strand cDNA. Finally, there is an isothermal, linear-amplification step using a second DNA/RNA chimeric primer, DNA polymerase, and RNase H in an isothermal reaction.
“The process repeats multiple times leading to the rapid accumulation of micrograms of amplified, single-stranded cDNA products,” Dr. de Feo explains. “The entire process takes approximately five hours.”
Two particularly important areas of application for NuGEN’s whole-transcriptome amplification technology include analyzing severely degraded FFPE specimens and analyzing highly-purified cell populations. “Clinical studies can last more than five years and cost hundreds of millions of dollars to conduct,” says Dr. de Feo. “However, now within three to six months, a researcher can take all those archived FFPE specimens and run whole-transcriptome analysis and quickly generate high-quality data.”
Another application is in the discovery environment where a small amount of target cells such as blood-cell subsets, sorted cells, or cells isolated by laser capture microdissection can be evaluated for gene-expression studies, Dr. de Feo adds.
Gene-expression profiling is increasingly adopting a systems biology approach. Data collection requires standardization of the tissue sample-preparation procedure, making stabilization of RNA, DNA, and proteins an absolute prerequisite for reliable analysis.
“Many labs are performing the analysis using the same sample,” says Vera Holländer, Ph.D., senior scientist at Qiagen. “It’s important to stabilize RNA, DNA, and proteins at the same time, because often there can be a lag time between harvest and extraction. This can change the biomolecular profile due to induction, degradation, etc.
“We recently released the Allprotect Tissue Reagent that can immediately and simultaneously preserve RNA and DNA as well as proteins in animal and human tissues. You can just drop a biopsy specimen into the solution and store it at a variety of temperatures for months without even needing to freeze the sample.”
The technology scores points for the all-important ability to preserve proteins in samples. “There are already some products for RNA stabilization on the market, such as our RNAprotect products that stabilize RNA in saliva and cells or PAXgene RNA Blood for RNA stabilization in whole blood, but there really weren’t alternatives for proteins,” according to Dr. Holländer. “An added benefit is that we also found that proteins can stay active when preserved in this way for subsequent functional analyses.”
Other advantages of the Allprotect technology, according to Dr. Holländer, are that, “It allows large numbers of samples to be easily processed. Also, it replaces inconvenient, toxic, and equipment-intensive methods such as snap-freezing of samples in liquid nitrogen, storage at -80°C, cutting and weighing on dry ice, and the need for immediate processing of harvested samples.”
This technology may be especially important for archived samples, reports Dr. Holländer. “We have performed tests such as RT-PCR, Western blot analysis, as well as mass spectrometry and showed that preserved tissue samples are quite stable. This presents a broad range of possibilities including saving samples for analysis by future technologies as they develop.”
The cost of sample preparation continues to increase. “This represents a large fraction of the analysis costs,” remarks Frederic Breussin, project manager for microfluidics, Yole Développement.
“We see a lot of development at the moment in automating sample preparation using microfluidics,” notes Breussin. “These technologies allow precise measurements using only a few microliters of sample for analysis. It’s the most efficient closed system, and operator error is eliminated.”
Microfluidics will likely have a major impact on point-of-care analysis, according to Breussin. “We believe the largest impact will occur for analyses in doctors’ offices, clinical labs, and even on the battlefield.
“Microfluidics have many uses in discovery, but before you see this technology appear on a larger scale, the industry will need to develop integrated, miniature, and low-cost instruments.” Microfluidics for diagnostic applications started in the late 90s. Nowadays, the supply chain is well established, which will allow lots of products to appear more quickly. For sample preparation, this means that you will see improvements in the near future that will enhance speed and sensitivity and at a reduced overall cost.”
Most experts agree that the area of RNA sample preparation, especially as it relates to drug discovery, is poised to solve most of its problems. For the future, as technologies expand and improve, they will easily meet the growing demand for high-quality, cost-efficient methods of RNA preparation.