Microarray-based comparison of three amplification methods for nanogram amounts of total RNA.
Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (One RA, Standard Protocol, or Two RA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 mug, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 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. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. Two RA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA.
Singh
Ruchira
R
Dept. of Anatomy & Physiology, College of Veterinary Medicine, Kansas State Univ., 1600 Denison Ave., Coles Hall 205, Manhattan, KS 66506, USA.
Maganti
Rajanikanth J
RJ
Jabba
Sairam V
SV
Wang
Martin
M
Deng
Glenn
G
Heath
Joe Don
JD
Kurn
Nurith
N
Wangemann
Philine
P
D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D046228:Microarray Analysis / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D012313:RNA