A robust method for the amplification of RNA in the sense orientation.
BACKGROUND
Small quantities of RNA (1-4 microg 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.


RESULTS
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.


CONCLUSIONS
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.
Marko
Nicholas F
NF
The George Washington University Medical Center, USA. nmarko@tigr.org <nmarko@tigr.org>
Frank
Bryan
B
Quackenbush
John
J
Lee
Norman H
NH
D000465:Algorithms; D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods*; D017931:DNA Primers; D003627:Data Interpretation, Statistical; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D015233:Models, Statistical; D021141:Nucleic Acid Amplification Techniques; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D016372:RNA, Antisense / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm
D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D016372:RNA, Antisense; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012313:RNA
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