Novel isothermal, linear nucleic acid amplification systems for highly multiplexed applications.
BACKGROUND
Global analysis of the genome, transcriptome, and proteome is facilitated by the recent development of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3'-Ribo-SPIA primes cDNA synthesis at the 3' polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthesis across the full length of the transcripts and thus provides whole-transcriptome amplification, independent of the 3' polyA tail.


METHODS
We developed isothermal linear nucleic acid amplification systems, which use a single chimeric primer, for amplification of DNA (SPIA) and RNA (Ribo-SPIA). The latter allows mRNA amplification from as little as 1 ng of total RNA. Amplification efficiency was calculated based on the delta threshold cycle between nonamplified cDNA targets and amplified cDNA. The amounts and quality of total RNA and amplification products were determined after purification of the amplification products. GeneChip array gene expression profiling and real-time PCR were used to test the accuracy and reproducibility of the method. Quantification of cDNA products (before and after amplification) at the 2 loci along the transcripts was used to assess product length (for evaluation of the 3'-initiated Ribo-SPIA) and equal representation throughout the length of the transcript (for evaluation of the whole transcript amplification system, WT-Ribo-SPIA).


RESULTS
Ribo-SPIA-based global RNA amplification exhibited linearity over 6 orders of magnitude of transcript abundance and generated microgram amounts of amplified cDNA from as little as 1 ng of total RNA.


CONCLUSIONS
The described methods enable comprehensive gene expression profiling and analysis from limiting biological samples. The WT-Ribo-SPIA procedure, which enables amplification of non-polyA-tailed RNA, is suitable for amplification and gene expression analysis of both eukaryotic and prokaryotic biological samples.
Kurn
Nurith
N
NuGEN Technologies, Inc.,Unit A, San Carlos, CA 94070, USA. nkurn@nugeninc.com
Chen
Pengchin
P
Heath
Joe Don
JD
Kopf-Sill
Anne
A
Stephens
Kathryn M
KM
Wang
Shenglong
S
D020869:Gene Expression Profiling / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
D012333:RNA, Messenger; D012313:RNA