Researchers from Applied Biosystems and the University of Ghent Demonstrate Megaplex™ miRNA Profiling Tools
News Nov 04, 2008
The study, entitled "High-throughput stem-loop RT-PCR miRNA expression profiling using minute amounts of input RNA," describes the simultaneous profiling of 450 human microRNAs from samples as small as a single cell. The paper appears online in the journal Nucleic Acids Research.
The study provides validation for a new workflow for miRNA analysis developed by Applied Biosystems to enable highly sensitive, high-throughput detection of miRNAs from extremely small biological samples.
MicroRNAs are a recently discovered class of non-coding small RNA molecules that are receiving attention from the scientific community for their important role in regulating gene expression and for recent research implicating these molecules in complex disease processes such as cancer.
Since the discovery of miRNAs, more than 800 human miRNAs have been identified and many more are awaiting experimental validation. In order to harness the potential of this important class of gene regulators, scientists require high-throughput and sensitive detection methods capable of measuring miRNA expression from very small biological samples, including single cells.
The purpose of the study was to test the ability of new tools based on Applied Biosystems' stem-looped, primer-based real-time PCR approach and industry-standard TaqMan® chemistry to accurately identify miRNAs in three different neuroblastoma cancer cell lines, both with and without a pre-amplification step.
In the study, sensitivity to single-cell levels was achieved using a novel approach incorporating an early-access version of the company's recently commercialized Megaplex™ Primer Pools.
Previous studies have reported the use of pre-amplification in combination with the stem-loop procedure; however, no published study thus far has evaluated the effects of the pre-amplification step on the fidelity of miRNA expression measurement. For that reason, one of the main goals of the study was to evaluate whether pre-amplification would compromise the accuracy of the results.
The researchers in this study reported unbiased and efficient pre-amplification efficiency, irrespective of miRNA expression level, using this method. In fact, pre-amplification actually enabled the detection of miRNAs that were undetectable in the experiments without pre-amplification, despite the use of 40-fold less starting material.
The Megaplex Primer Pools consist of the Megaplex RT Primers and the Megaplex PreAmp Primers. The Megaplex™ RT Primers are a set of highly multiplexed reverse transcription (RT) primers that greatly reduce the number of RT reactions required, thereby streamlining the workflow and reducing the sample input required to create a comprehensive miRNA profile.
Megaplex™ PreAmp Primers, used in conjunction with the TaqMan® PreAmp Master Mix, enable amplification of the starting RNA sample prior to performing detection using real-time PCR, significantly increasing sensitivity when working with limited samples.
"The high specificity and sensitivity of this method makes it ideal for high-throughput miRNA expression profiling," said Jo Vandesompele, Ph.D., professor at Ghent University Hospital. "This novel technology from Applied Biosystems opens the way for profiling the miRNAome from small cell populations or individual cells."
Other technologies enable profiling of multiple miRNAs in a single experiment, but such approaches generally require significant amounts of input RNA - 100ng or more - and therefore preclude the use of very small clinical biopsies or analysis of small subsets of cells or single cells. This limits researchers' abilities to conduct important experiments for diseases such as cancer in which often only minute samples are collected, or originate from archived formalin fixed paraffin embedded samples in which the genetic material may be degraded and RNA yield is relatively low.
"The ability to detect and accurately quantify all known miRNAs from a few cells or even a single cell with these new tools enables researchers to look further and deeper into the important role of miRNAs in living systems," said Caifu Chen, Ph.D., scientific fellow at Applied Biosystems. "We expect that researchers will now be able to identify novel biomarkers for complex diseases such as cancer, as well as better understand how miRNAs regulate development of a single neuron or embryo."
The Megaplex miRNA tools used in this study are now commercially available from Applied Biosystems for use with the TaqMan® MicroRNA Arrays and with individual TaqMan® MicroRNA Assays. Together, these tools are expected to streamline miRNA analysis workflows for life science researchers and provide comprehensive coverage of miRNA expression in small biological samples or single cells.