Submitted on May 25, 2004
Accepted on December 17, 2004
Microarray based comparison of three amplification methods for nanogram amounts of total RNA
Ruchira Singh1, Rajanikanth J Maganti1, Sairam V Jabba1, Martin Wang2, Glenn Deng2, Joe Don Heath2, Nurith Kurn2, and Philine Wangemann1*
1 Anatomy & Physiology, Kansas State University, Manhattan, KS, USA
2 NuGen Technologies, San Carlos, CA, USA
* To whom correspondence should be addressed. E-mail: wange{at}vet.k-state.edu.
Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples available from microdissection, laser-capture microscopy or needle biopsies. A novel system, based on Ribo-SPIATM technology, has recently been introduced (RS, Ovation-Biotin amplification and labeling system, NuGEN Technologies, San Carlos, CA). The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS, NuGEN Technologies), and two T7-based systems involving one or two rounds of amplification (OneRA, Standard Protocol, Affymetrix, Santa Clara, CA or TwoRA, Small Sample Prototcol, Version II, Affymetrix) were evaluated. Mouse kidney (MK) and universal reference (MUR) RNA samples, 0.3 ng to 10 µg, were analyzed by high-density Affymetrix Mouse 430 2 GeneChip® arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold-increase necessary for significance, were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10 ng RNA samples. 24 of 26 genes verified by RT-PCR were detected in samples prepared by pRS. TwoRA yielded somewhat higher call concordances than RS and pRS (91.8% versus 89.3% and 88.1%, respectively). In conclusion, although all target preparation methods were found suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng total RNA. In addition, RS and pRS were faster and simpler than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA.
