Linear RNA Amplification For Expression Analysis

NuGEN Technologies, Inc.

821 Industrial Road, Unit A
San Carlos, CA, 94070
Website: http://www.nugeninc.com

Linear RNA Amplification For Expression Analysis

by Nurith Kurn, Ph.D. and Joe Don Heath, Ph.D.(s)

click the image to enlarge

Linear amplification
RNA amplification is rapidly becoming a routine and necessary step when preparing samples for expression analysis— particularly when using microarrays. RNA amplification provides the sensitivity to detect expression differences of low abundance transcripts that is critical to obtaining a complete understanding of the biology of a given system. Enhanced sensitivity is also necessary for gene expression analysis of more biologically-significant, homogeneous samples that are often comprised of low numbers of cells (e.g., microdissected biopsies, fine-needle aspirates, sorted cells, and embryonic structures). Additionally, recent publications have shown that routine RNA amplification actually improves the quality of microarray results by providing more array-to-array consistency (Feldman 2002, Polacek, 2002). Finally, the utility of RNA amplification is not limited to microarray studies. Although quantitative PCR-based methods (Q-PCR) do not require the amount of starting material that microarrays do, sample amounts are often limiting when trying to quantify the expression levels of many genes in a panel of Q-PCR reactions. A "pre-amplification" of mRNA that maintains the relative transcript amounts of the original sample addresses this issue.

Although the benefits of routine amplification — more reliable and reproducible results — are readily evident, RNA amplification of samples intended for expression analysis is often avoided. Primarily, this avoidance occurs because the amplification step can be a very time-consuming and tedious process. Further, amplification of only a portion of the samples in a given study can lead to significant data problems. Differences in sensitivity, enzyme activities, labeling efficiencies, and amplification variability between procedures can make comparison of results from samples prepared by two different methods highly problematic. In addition, there are resource and logistical issues associated with maintaining several preparation procedures in a single laboratory. Finally, standardized laboratory practices are important to ensure proper and consistent sample handling. These practices can be difficult to implement when more than one methodology is used to prepare samples for analysis. To avoid these issues and facilitate the routine amplification of all samples both large and small NuGEN Technologies Inc. has developed RNA-based single-primer isothermal amplification (Ribo-SPIA) technology that delivers high fidelity, sensitive RNA amplification for gene expression analysis.

Ribo-SPIA technology
As opposed to exponential PCR amplification, the Ribo-SPIA method is linear, which means it maintains the relative representation of each transcript species present in the original sample (see Figure 1). This feature is critical for any process used to prepare samples for expression analysis. Ribo-SPIA technology is also distinctly different from methods that use in vitro transcription with a T7 polymerase. Unlike T7-based methods, a single Ribo-SPIA amplification typically provides ample material for multiple hybridizations, even from just a few nanograms of total RNA. By starting with as little as 5 nanograms of total RNA, the Ribo-SPIA technology preserves limited sample sources and reduces the cost and difficulty of sample procurement. For very small samples, T7-based methods require multiple amplification cycles which create procedural complexity, require additional days of processing, and introduce representational bias. In addition, varying the number of amplifications results in differential treatment of different-sized samples, thus creating logistical problems and making it difficult to compare results from sample-to-sample. Designed as a rapid, single-day, add-and-incubate method for routine preparation of samples for expression analysis, Ribo-SPIA technology provides a standardized process to maximize sensitivity and reproducibility.

Ribo-SPIA technology differs from T7-based approaches by utilizing a chimeric RNA/DNA primer comprising a known RNA sequence tag on the 5'-end of an oligo-dT cDNA primer (see Figure 2). Incorporation of this chimeric DNA/RNA primer means that, after first and second strand synthesis, the resulting double-stranded cDNA contains a short RNA/DNA heteroduplex segment of known sequence at the 5' end of the first strand. It is this unique RNA/DNA heteroduplex at the end of the double-stranded cDNA that provides the basis for the linear amplification (i.e., SPIA) that makes this procedure distinctive.

The SPIA reaction is initiated by incubating with RNaseH, a second chimeric DNA/RNA SPIA primer, and DNA polymerase. RNaseH only digests RNA that is hybridized to DNA, so it digests the short RNA segment leaving the complementary cDNA portion exposed. Exposure of this complementary sequence allows the SPIA primer to hybridize to its complementary sequence and recruit the polymerase to initiate synthesis of another cDNA strand, which displaces the original strand. A continuous cycling of RNaseH degradation, primer binding with extension, and displacement of a portion of the previous cDNA enables rapid linear production of thousands of replicates from each cDNA template.

Unlike T7-based processes that cycle between DNA and RNA, Ribo-SPIA technology uses DNA polymerase to replicate cDNA, which produces a very stable and archivable single-stranded cDNA product. The stable cDNA product minimizes the risk of false gene expression differences that can result from non-specific degradation of labeled RNA during preparation and storage.

To initiate the SPIA cycle after cDNA synthesis, reagents are simply added to the mix. No purification is required through the process — from cDNA synthesis through amplification. This simple and rapid add-and-incubate process minimizes the possibility of technical errors, prevents the loss of material during purification, and makes the whole procedure easy to automate.

Amplification and labeling systems
NuGEN has commercialized three products powered by Ribo-SPIA technology: the Ovation Aminoallyl System, the Ovation Biotin System, and the Ovation RNA Amplification System. The first two kits have been optimized to amplify and label RNA samples for hybridization to spotted or Affymetrix GeneChip microarrays, respectively. The third system preamplifies mRNA for use in QPCR and other gene expression experiments. Each Ovation System is based on the same Ribo-SPIA technology, making it appropriate for laboratories that utilize multiple platforms. Thus, regardless of the sample size or platform, researchers can standardize on a single, one-round, add-and-incubate amplification for sample preparation.

The Ovation Aminoallyl System prepares samples for hybridization to standard oligonucleotide microarrays spotted on glass slides. This kit incorporates an aminoallyl-label during Ribo-SPIA amplification, so the cDNA product produced with this system can be readily conjugated to cyanine dyes for detection purposes. This indirect incorporation of cyanine dyes is preferable to direct incorporation of cyanine-labeled nucleotides. Direct incorporation of cyanine dyes can produce differential signal artifacts for some transcripts since different cyanine-labeled nucleotides are incorporated at different rates. With the indirect aminoallyl conjugation used in the Ovation System, dye representation is very consistent from sample to sample, regardless of which cyanine dye is used.

The Ovation Biotin System is designed to specifically prepare samples for hybridization to Affymetrix GeneChip arrays. As opposed to an aminoallyl label, this system incorporates a biotin label using a proprietary approach that includes chemical coupling of biotin to fragmented cDNA amplification products. This approach has been validated on Affymetrix GeneChip arrays using a variety of sample types, including laser microdissected samples (see Figures 3 and 4).

The Ovation RNA Amplification System preamplifies mRNA prior to Q-PCR analysis. The system expands mRNA from potentially limited, biologically important samples such as flow sorted cells or small biopsies, giving researchers the ability to both analyze all gene transcripts of interest and to archive additional material for future reference.

The Ribo-SPIA technology addresses the need for a sensitive RNA amplification process that can be routinely used in the preparation of sample for gene expression analysis. The Ovation Systems enable researchers to generate more reliable and reproducible results — even from low abundance transcripts — which leads to more meaningful discovery.

About the authors
Nurith Kurn, Ph.D., is co-founder and CSO, and Joe Don Heath, Ph.D., is director of technical services at NuGEN Technologies (San Carlos, CA). For more information, contact Joe Don Heath. Email: jheath@nugeninc.com. The authors wish to acknowledge the contributions of NuGEN's scientists.