March 1, 2009 (Vol. 29, No. 5)
K. John John Morrow Jr. Ph.D. President Newport Biotech
New Methodologies Are Being Counted On to Bring Order and Repeatability to Gene Expression
In recent years, drug discovery has become a search for targets that take the form of gene-expression differences between normal and disease states. One of the leading approaches has been based on building gene-expression profiles derived from microarray analysis. In such investigations RNA samples from different tissues, organs, disease states, and individuals are evaluated against microarrays consisting of large numbers of gene fragments. While this strategy is widely used, initially results were inconsistent, they could not be duplicated, and putative markers failed to survive critical assessment.
One reason for the failure of such studies was the poor quality of the RNA preparations, given that this molecule is easily degradable and stringent precautions must be met in its isolation. But recently, a number of suppliers have developed approaches to ensure high-quality RNA isolation.
Qiagen offers a variety of products for the molecular biologist, including kits to ease the challenges of RNA purification, according to Barry Westfall, regional marketing manager for North America. Qiagen’s purification kit for nucleic acids and proteins goes by the trade name AllPrep, which includes the stabilization reagent, also available as a free-standing product.
The RNeasy Mini Kit was designed to process up to 100 µg total RNA from mammalian cells, tissues, and yeast. These kits are based on the use of disposable modules with silica membranes, which become attractive to the nucleic acids because of the buffer systems.
All the companies surveyed for this article offer alternatives to the phenol/chloroform extraction procedure, a venerable warhorse whose use goes back to the early days of molecular biology in the 1960s. Today there is great pressure on both academic and commercial laboratories to lower their contribution to the contamination of the water supply to zero.
However, the principle driver for new methodologies has been the need for a technique that could deliver high-quality total RNA in minutes. Moreover, since researchers aim to analyze samples smaller by orders of magnitude than those that they would have attacked 20 years ago, Qiagen has designed technologies that can derive RNA, DNA, and protein all from the same microsample.
This reconfiguration means that Qiagen can now deliver products that generate ready-to-use RNA in yields from very small to large amounts of starting material. Their miRNeasy and miScript products allow preservation of all the miRNA in a sample. Thus the RNA molecules are all purified and material can be scanned when a new miRNA is discovered.
Qiagen also offers a number of robotic equipment options to move processing of samples forward at an accelerated pace. The QIAsymphony SP is a compact integrated system, with built-in touchscreen and computer designed to operate an automated system with flexible processing of a wide range of samples. It can handle up to 96 samples, in batches of up to 24 per run, with sample volumes up to 1 mL.
The system is designed for automated purification of nucleic acids or 6xHis-tagged recombinant proteins from a range of sample types. The platform is easy-to-use with prefilled reagent cartridges that permit flexible processing of 1 to 96 samples per run. The use of bar code reading enables full sample and reagent tracking. An additional product is FastLane, allowing qPCR straight from cells, including SYBR Green, Probe, or Multiplex probe qPCR.
In addition, Qiagen has embarked upon a partnership with Leica Microsystems to produce a laser microdissection and nucleic acid purification tool. The system can cut discrete portions of a tissue section with great precision; the excised portion is collected by gravity feed into a tube and can then be analyzed for specific DNA or RNA content. The technique favors the use of flash-frozen cryosections to avoid formalin and produce the highest quality nucleic acid molecules.
“Quality, yield, and consistency are the three guiding principles of researchers in the field of RNA analysis,” explains Brian Kim, director for Ambion Technologies at Life Technologies. This means that, from the inception of a procedure, RNA profiles must be locked down. “The samples that investigators are targeting are heterogeneous, all different from one another,” he continues.
Obtaining high-quality, intact RNA is the first and often the most critical step, and to this end Ambion produces a range of products. “To meet these needs, we produce kits for processing blood, paraffin-embedded tissue, cultured cells, and material from different organs,” he says.
A long-standing product in the Ambion line is the RNAlater® Tissue Storage:RNA Stabilization Solution that allows the researcher to postpone RNA isolation indefinitely after tissue collection without sacrificing the integrity of the RNA. Dissected tissue or collected cells are dropped into the RNAlater solution that permeates the cells, stabilizing the RNA, and allowing biological materials to be stored for an indefinite period.
The next stage of RNA processing, RNA isolation, is advanced by kits for total or poly(A) RNA isolation compatible with a range of plants, microorganisms, and animals. TRI Reagent® is a single, phenol-based, homogenous solution containing a combination of denaturants and RNase inhibitors, used in a single-step disruption and separation procedure. Another approach is the RNAqueous® Technology, a rapid, filter-based RNA isolation system that does not require the use of phenol, chloroform, or other toxic organic chemicals.
Alternatively, total RNA may be isolated using Ambion’s ToTALLY RNA™ Kit, an alternative to the guanidinium thiocyanate/acid phenol:chloroform method but modified to reduce DNA, carbohydrate, heme, and other contaminants that can foul the preparations.
The company’s products have features that make them especially useful for bacterial RNA purification, a task which presents its own idiosyncrasies. Speed is critical in the purification of prokaryotic RNA due to the short half-life of bacterial mRNA and the need to rapidly “freeze” the mRNA expression profile. Easily lysed gram-negative bacteria may be pipetted directly into a boiling lysis buffer of choice (without even removing the culture medium), and RNA can be immediately extracted with the TRI Reagent.
One of Ambion’s leading products, according to Kim, is the RecoverAll™ Total Nucleic Acid Isolation Kit for extraction of total RNA or DNA from formalin- or paraformalin-fixed, paraffin-embedded tissues. This kit enables the molecular analysis of archived tissue samples at both the genomic and gene-expression levels and is suitable for qRT-PCR, mutation screening, and microarray analyses. The four-hour protocol yields RT-PCR-competent RNA even for rare messages, Kim says.
Eukaryotic mRNA is used for the construction of random-primed cDNA libraries. Removal of ribosomal and transfer RNA produces a 30-fold enrichment of a specific message. Ambion’s Poly(A)Purist™ Kits are designed for the isolation of the high-purity mRNA from total RNA, without sacrificing yield. The Poly(A)Purist and MicroPoly(A)Purist Kits include premeasured aliquots of oligo(dT) cellulose, whereas the Poly(A)Purist MAG Kit incorporates oligo(dT) magnetic bead-based purification.
Isolation of mRNA from bacteria presents a special challenge, as they lack the relatively stable poly(A) tails found on eukaryotic messages. The MICROBExpress™ Bacterial mRNA Isolation Kit is designed to remove >95% of the 16S and 23S rRNA from total RNA of E. coli and other bacterial species.
The kit is suitable for rapid mRNA purification from a broad spectrum of gram-positive and gram-negative bacteria. In the first step of the MICROBExpress procedure, bacterial total RNA is mixed with an optimized set of capture oligonucleotides that bind to the bacterial 16S and 23S rRNAs. Next, the rRNA is removed from the solution using derivatized magnetic microbeads. The mRNA remains in the supernatant and is recovered by ethanol precipitation.
Perhaps one of the most striking features of the RNA-isolation kits is their speed, in which whole segments of traditional isolation protocols are eliminated, and a molecule, ready for amplification, can be isolated in minutes. Examples include the Power SYBR® Green and Fast SYBR® Green Cells-to-CT™ Kits that employ a novel cell lysis and RNA-stabilization technology, according to Kim.
This strategy completely eliminates the need for laborious and time-consuming RNA purification and integrates the lysis technology into a complete workflow including reverse transcription reagents and high-performance Power SYBR Green or Fast SYBR Green Master Mixes.
Until recently, small RNAs were looked upon as a distracting contaminate, yet they are now recognized as critical features of the regulatory process. “Our mirVana™ miRNA isolation kit enables cancer researchers to isolate total RNA that contains small RNAs such as miRNA, siRNA, and snRNA that are often lost during traditional RNA isolation methods,” Kim states.
“Our exclusive focus on oncology requires us to deal with a large number of samples for RNA profiling in microarrays and other large-scale platforms,” says Stephen Tirrell, Ph.D., director of molecular technologies at Millennium Pharmaceuticals, The Takeda Oncology Company.
“Our biggest challenge is maximizing the quality and availability of biopsy samples from the trial site,” Dr. Tirrell explains. “Getting a good biopsy from the clinic to the lab is a monumental task, demanding an all-out effort. In oncology, tumor content of the biopsy is a big variable that adds to the challenge.”
Dr. Tirrell feels that new technologies developed for RNA isolation and purification have greatly helped to move the field forward. “The new kits are much faster and more reliable,” he states. “Before they were available we could spend a week and a half just processing the samples and isolating the material that was required to do the actual experiments.”
A component of cancer biomarker studies is focused on a search for cancer-related biomarkers in whole blood, and much of Dr. Tirrell’s investigations are based on this approach. However blood has its limits in such investigations. “It is a good surrogate tissue for biomarker studies, however it does not directly inform you what is happening in the tumor,” he adds.
Amplification of Tiny Samples
“Our technology focuses on minute amounts of RNA such as those from small cell populations, microdissection samples, fluorescence-activated cell-sorting samples, or fine-needle aspiration biopsies,” says Jeffrey Hung, Ph.D., director of marketing at SABiosciences.
The company has introduced its RT2 Nano PreAMP cDNA Synthesis Kit, designed to take on 1 ng total RNA on PCR arrays. Using multiplex PCR-based preamplification it provides amplification of gene-specific cDNA target templates with minimal bias. With this kit one can prepare enough cDNA from each RNA sample for gene-expression analysis on as many as four different pathways, Dr. Hung says. A built-in external RNA control allows the detection of inhibitors of reverse transcription, ensuring the efficiency of the first-strand cDNA synthesis reactions.
“The beauty of the technology is that it enables the accurate and sensitive analysis of up to four different pathway PCR Arrays (~350 genes) from as few as 100 cells or 1 ng total RNA, Dr. Hung adds. For precious samples that can only be obtained in minute amounts, we provide an ideal solution for pathway gene-expression analysis.”
RNA has always presented challenges to molecular biologists, and it is clear that large-scale projects involving isolation and characterization of thousands of gene products will require drastic improvements in RNA-handling technology. The search for drug targets has adopted microarray technology based on gene expression. However the history of the last decade of genomics and proteomics analysis is not encouraging for the data-without-a-model concept of doing science.
For years, pharmas and biotechnology companies and research labs cranked out results from genomic and proteomic screening, in which they searched for viable targets for new therapies. After spending millions of dollars and countless years of effort they came up with essentially nothing. The work was not well controlled, it was not thoroughly researched, the experimental designs were poor, and there was no hypothesis or model, just an endless search for targets.
There were many papers in the peer-reviewed literature as well as countless reports at meetings in which hundreds or thousands of new potential targets were described. But these all vanished when they could not be confirmed or when drugs that attacked these targets were found to have serious side effects.
The new technologies now available provide a much-needed solution to the problem. They are simple, robust, and repeatable, and the validation process removes unnecessary steps.
Until recently, lab workers would question the cost of a kit since they had all those reagents on the shelf. Yet the consistency, simplicity of use, and lack of error in their assembly makes them well worth the additional cost.
These products will go a long way toward bringing order and repeatability into the field of gene expression, and should speed the process of translational medicine through good, reliable data.