Increased sample throughput on Applied Biosystems genomic analysis platform simplifies study of small RNAs
30 Jul 2008

Research related to the roles that small non-coding RNA molecules play in living cells is one of the fastest growing areas in biology. For these kinds of studies, researchers analyse large numbers of biological samples to examine how patterns of differentially expressed RNAs influence cellular processes related to basic biology and complex diseases such as cancer. To enable scientists to rapidly discover and profile novel small RNA molecules without requiring prior knowledge of sequence information, Applied Biosystems today announced the commercial availability of a new small RNA expression analysis solution optimised for the SOLiD™ System, the company’s ultra-high throughput platform for DNA sequencing and genomic analysis research. This solution includes a multiplexing technology (also known as barcoding) that enables the simultaneous analysis of up to 256 biological samples in a single sequencing run. The multiplexing technology is currently available in limited release through an early access programme.

This new multiplexing technology is expected to simplify the workflows and reduce the experimental time and cost of applications such as studying the function of small RNA molecules, validation of whole genome association results, targeted resequencing, chromatin immunoprecipitation (ChIP), rare and somatic mutation detection, and epigenetic studies.

Use of this multiplexing technology is expected to dramatically reduce both labour and reagent costs for researchers preparing samples for the SOLiD System by allowing them to pool multiple libraries of nucleic acid fragments, which reduces the number of emulsion PCR reactions. These pooled samples can then be sequenced in a single reaction. The combination of the SOLiD System’s high sample and sequence throughput allows researchers to use each pooled, labelled set of samples in different slide configurations, enabling the ability to sequence 256 samples in a single sequencing run.

The new SOLiD Small RNA Expression Kit provides researchers with an innovative workflow that greatly reduces the time, cost and experimental variability associated with small RNA library preparation. The SOLiD System is capable of generating up to 240 million sequence reads, or tags per run, for RNA expression analysis applications. The kit converts total RNA into a library suitable for input into the SOLiD System workflow for high throughput genomic analysis. The simple protocol reduces the library sample preparation workflow to a single day compared to four days with alternative platforms. The kit is expected to improve the detection of biologically relevant changes for researchers working with small RNA molecules. It also requires very small sample quantities, enabling studies that were not previously possible.

One participant in the early access programme for multiplexing technology in expression analysis applications is Dr Jesse M Gray, postdoctoral research fellow in the laboratory of Dr Michael E Greenberg, director of the F M Kirby Neurobiology Center at Children’s Hospital Boston. For many years, the Greenberg laboratory has been investigating how gene expression changes in response to nerve cell activation. Through whole transcriptome analysis using the small RNA expression kit with multiplexing capability, Dr Gray has been able to detect and characterise not just whether neuronal activity induces a gene to be expressed, but which variants of that gene are expressed.

“Before we had the multiplexing technology, preparing multiple samples for a transcriptome analysis experiment was time intensive. Currently, use of the SOLiD System multiplexing technology, which allows us to pool 16 samples in one sequencing run, has enabled us to complete the same analysis in one week at a five to 10-fold saving in reagents used for the emulsion PCR step,” said Dr Gray. “By using this approach, it is now feasible to include not just one or two, but many different samples or conditions in a sequencing run. This kind of breakthrough on the SOLiD platform will bring us one step closer to making a major shift away from array-based to sequencing-based gene expression assays.”

Use of the new small RNA expression analysis solution with multiplexing technology will allow researchers to label individual biological samples with a unique identifier when preparing nucleic acid fragment libraries for tag-based applications, such as RNA expression analysis. By enabling researchers to simultaneously investigate the RNA contents of multiple samples, this technology harnesses the ultra-high throughput capabilities of the SOLiD System.

One class of small non-coding RNAs that has been studied extensively in recent years is microRNAs, which are gene expression regulatory molecules comprised of about 20 nucleic acid bases. Approximately 6,200 microRNAs have been discovered across organisms, but advances in technology such as the SOLiD System are expected to reveal many more novel molecules of potential scientific interest. Because microRNAs consist of just a few bases, effective profiling of samples for these small RNAs will benefit from a multiplexing technology that allows researchers to investigate greater numbers of samples that require a lower throughput of sequence reads per sample than do large scale genomic analysis studies.

In addition to the multiplexing technology, the SOLiD System’s 2-base encoding benefits microRNA profiling studies. In profiling of microRNAs, sequencing allows researchers to discover variants that may occur in different species of these molecules, which can greatly influence the function of the microRNA molecule. For discovery of genetic variants, highly accurate sequencing is necessary for reliable data. The SOLiD System’s 2-base encoding chemistry discriminates random or systematic errors from true variants throughout the genome to reveal genetic variants with greater than 99.94 per cent accuracy.

“As scientists learn more about the key roles that small RNAs play in biological systems, the SOLiD platform will be integral to enabling new approaches for gene expression analysis,” said Shaf Yousaf, president for Applied Biosystems’ molecular and cell biology genomic analysis division. “This new small RNA analysis kit, equipped with multiplexing capability, will accelerate these research studies and enable them to be conducted in a more efficient and cost-effective manner.”

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