by Caitlin Smith
Although microarrays are an essential microRNA (miRNA) screening tool for many, another tool with a wider dynamic range is gaining on them—real-time PCR. Also known as quantitative PCR (qPCR), this method enables a quantitative, real-time analysis of microRNA expression—one that is still improving in many aspects. “The most important characteristics of a microRNA qPCR assay a researcher should look for are sensitivity, specificity, robustness and thereby accuracy, ease of use and duration of protocol,” says Peter Mouritzen, R&D director for life science at Exiqon. “In addition, a proper design of the study initially is also very important.” Addressing all these concerns simultaneously is a tall order. Here we’ll consider some of these issues in the context of current tools in qPCR-based microRNA analysis.
Do you need microRNA high-throughput profiling capabilities?
A useful tool for screening studies is the high-throughput profiling panel. Life Technologies recently released its TaqMan® OpenArray® Human MicroRNA Panel for high-throughput profiling of human microRNAs and the TaqMan OpenArray Rodent MicroRNA Panel for high-throughput profiling of mouse and rat microRNAs. The panels can run up to three samples each, with six logs of dynamic range. “For high-throughput microRNA profiling, researchers are time-crunched, and their customers and research demand accurate and affordable answers to get to validation quickly,” says Dennis Fantin, product management leader for qPCR at Life Technologies. “Unlike hybridization-based microRNA profiling platforms, TaqMan OpenArray MicroRNA Panels yield the highest concordance to the most commonly used tool for validation—individual TaqMan MicroRNA Assays. Utilizing nanoliter fluidics, TaqMan OpenArray MicroRNA Panels provide researchers with the fastest, easiest and most affordable path to validated data.” In addition, Life Technologies offers tools for low- to medium-throughput profiling with its TaqMan Array MicroRNA Cards and TaqMan MicroRNA Assays.
With a 3 hour short protocol Exiqon also seeks to address researchers needs for running high-throughput profiling. To aid overall throughput , Exiqon and MultiD released a new version of their qPCR data analysis software, GenEx, in June. “The software has greatly improved user-friendliness, with an interactive wizard taking the user through the initial steps of the data import, selection of proper reference genes for normalization and final data analysis in a few . . . simple clicks,” says Mouritzen. “However, the most important improvement of the new GenEx version is the ability to support high-throughput data analysis, where hundreds of qPCR data files can be imported in only a couple of minutes.”
How many microRNAs are you interested in?
Are you interested in detecting single microRNAs or hundreds of different microRNAs? Various tools are available for these different types of applications. Qiagen plans to launch its new miScript II PCR System for microRNA analysis in October. This system, which integrates Qiagen’s previous offerings with those of recently acquired SABiosciences, includes a reverse transcription kit, a SYBR Green PCR kit, arrays for microRNA profiling and individual assays to detect mature and precursor microRNAs or other non-coding RNAs. “One key benefit of Qiagen’s miScript PCR System is that it allows detection of hundreds of miRNAs from a single cDNA preparation,” says Constanze Kindler, senior global product manager at Qiagen. “This is in contrast to other systems, where scientists have to process one reverse transcription and PCR reaction for each miRNA they are interested in. The miScript II RT Kit is also the only one on the market that enables scientists to generate cDNA according to their requirements.” She adds that “researchers now have the choice between two specialized buffers for cDNA generation,” and explains that one buffer can be used to perform miRNA-specific reverse transcription with an extremely low background (ideal for screening), and that the other is compatible with more universal reverse transcription (to analyze miRNA, pre-miRNA and mRNAs).
Do you need to focus on one disease or pathway?
Qiagen offers the convenience of PCR arrays that focus miRNA analysis on particular diseases or pathways. “We offer up-to-date miRNomes for human, mouse, rat and dog for all available real-time cyclers, [and] also fully customizable miRNA PCR arrays,” says Kindler. “One example is an miRNA array that is dedicated to profiling circulating miRNA from plasma or serum, which works tremendously well with an optimized protocol for miRNA isolation from plasma and serum. The addition of a synthetic miRNA as a spike in control enables data normalization.”
Exiqon also offers focused panels to help researchers narrow their research questions. In August 2011, Exiqon released the first in a series of microRNA Focus PCR Panels. “The focus panels are collections of qPCR assays for microRNAs believed to have a central role in specific diseases, development, cells or body fluids,” says Mouritzen. “Exiqon has leveraged extensive knowledge gathered from diagnostic programs into making a selection of microRNAs for a Cancer microRNA Focus PCR Panel and a Serum/Plasma microRNA Focus PCR Panel. We are convinced that these Focus PCR Panels will greatly improve microRNA qPCR analysis in many research fields, and we look forward to releasing more validated focus panels for other research areas in the coming months.”
Do you need high sensitivity?
If your experiments require very high sensitivity, locked nucleic acids may further your research. Exiqon’s microRNA qPCR assay design is based on its proprietary Locked Nucleic Acid (LNA™) technology, which delivers high sensitivity. “The majority of Exiqon’s assays detect down to an equivalent of only five to 10 microRNA copies in the PCR,” says Mouritzen. “The high sensitivity is obtained by applying Exiqon’s proprietary LNA™ technology in PCR primers and through a highly efficient cDNA synthesis reaction that universally reverse transcribes all microRNAin a single cDNA preparation. The incorporation of LNA™ in the primers increases primer melting temperature, allowing shortening of PCR primers to facilitate design of two microRNA-specific PCR primers per microRNA, rather than only one microRNA-specific primer, as seen in competitive systems.”
LNA™ technology enables higher sensitivity by increasing primer affinity for their target. “The challenge in developing a sensitive and specific qPCR application for microRNA is the short length of microRNAs,” says Mouritzen. “Two microRNA-specific PCR primers per microRNA in theory should provide better specificity than one, and this is also what is observed in reality. The fact that LNA-based primers can be made shorter allows for making two microRNA-specific PCR primers that do not overlap, which thereby [reduces] assay background [noise].”
The sensitivity that you require also may depend on the type and quantity of your sample. “Some biological samples, like blood serum and plasma, contain very low amounts of microRNA and other nucleic acids,” says Mouritzen. “The enhanced sensitivity of Exiqon microRNA qPCR assays enables robust profiling of all microRNAs represented on Exiqon Human Panel I, using just 35 µL of serum or plasma. High sensitivity in assays is of particular importance for serum/plasma samples, [because] use of more sample will not provide for better detection of the microRNAs, as one would think. If applying larger sample sizes, one will increase the level of RNA inhibitors, which will harm the qPCR reaction.”
Improvements for the future
Real-time PCR-based microRNA analysis will become even more useful as improvements are made in the near future. For example, Fantin notes that there’s room for improvement with low sample volumes: “The need for customers to profile microRNAs when sample is limiting—
FFPE [formalin-fixed paraffin embedded], LCM [laser capture microdissection], single cell and sub-cellular fractions—has provided unique challenges and new opportunities to develop solutions around low-sample-input microRNA profiling.” Fantin also believes performance generally needs to improve. “Many real-time PCR-based microRNA concepts only use a single microRNA-specific primer, and in most of these concepts this primer contains sequence corresponding to the full length of the microRNA,” says Fantin. “Assays based on full-length, microRNA-identical primers are prone to give background. In fact, to many scientists it is conceptually wrong, for contamination reasons, to introduce a DNA oligo in the PCR workflow which is identical to the target sequence you wish to amplify.”
Another area for improvement is low-GC-content targets. “A drawback of many microRNA real-time PCR concepts is poor sensitivity when the target microRNA has low GC content,” says Fantin. Both Fantin and Mouritzen agree that since more than 20% of known microRNAs have a GC content below 40%, and given the fact that microRNAs are only about 22 nucleotides long, it is impossible to design standard DNA PCR primers that have a melting temperature compatible with normal PCR cycling conditions. Even standard PCR primers can have difficulty discriminating between members of microRNA families, which can differ by only one or a few nucleotides.
Mouritzen notes that LNA™ technology can improve the prospects for low-GC targets. “The use of two shorter LNA™-based primers not only results in high assay sensitivity for all microRNAs, independent of their GC content, but it also provides assays with a very high level of specificity,” says Mouritzen. “MicroRNAs with low GC content are difficult to design primers for, as they will have low melting temperatures,” meaning they will suffer from poor specificity and sensitivity. “But a higher melting temperature can be achieved by adjusting the content and placement of LNA™ molecules, thereby increasing the assay performance.” Though important details remain to be ironed out, qPCR-based microRNA analysis is a powerful technique that will continue to grow.