A robust method for the amplification of RNA in the sense orientation
1 The George Washington University School of Medicine
2 The George Washington University Medical Center, Department of Biochemistry and Molecular Biology
3 The George Washington University Medical Center, Department of Pharmacology
4 The Institute for Genomic Research (TIGR), Dana-Farber Cancer Institute, Department of Biostatistics and Computational Biology and Harvard School of Public Health, Department of Biostatistics
BMC Genomics 2005, 6:27 doi:10.1186/1471-2164-6-27Published: 1 March 2005
Small quantities of RNA (1–4 μg total RNA) available from biological samples frequently require a single round of amplification prior to analysis, but current amplification strategies have limitations that may restrict their usefulness in downstream genomic applications. The Eberwine amplification method has been extensively validated but is limited by its ability to produce only antisense RNA. Alternatives lack extensive validation and are often confounded by problems with bias or yield attributable to their greater biological and technical complexity.
To overcome these limitations, we have developed a straightforward and robust protocol for amplification of RNA in the sense orientation. This protocol is based upon Eberwine's method but incorporates elements of more recent amplification techniques while avoiding their complexities. Our technique yields greater than 100-fold amplification, generates long transcript, and produces mRNA that is well suited for use with microarray applications. Microarrays performed with RNA amplified using this protocol demonstrate minimal amplification bias and high reproducibility.
The protocol we describe here is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of microarray platforms, such as cDNA arrays, oligonucleotide arrays, and Affymetrix GeneChip™ arrays.