Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA - PubMed (original) (raw)
doi: 10.1038/nbt729. Epub 2002 Aug 12.
Affiliations
- PMID: 12172558
- DOI: 10.1038/nbt729
Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA
Norman N Iscove et al. Nat Biotechnol. 2002 Sep.
Abstract
Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification methods could be used in these cases, but it is difficult to amplify different transcripts in a sample without distorting quantitative relationships between them. Linear isothermal RNA amplification has been used to amplify as little as 10 ng of total cellular RNA, corresponding to the amount obtainable from thousands of cells, while still preserving the original abundance relationships. However, the available procedures require multiple steps, are labor intensive and time consuming, and have not been shown to preserve abundance information from smaller starting amounts. Exponential amplification, on the other hand, is a relatively simple technology, but is generally considered to bias abundance relationships unacceptably. These constraints have placed beyond current reach the secure and routine application of microarray analysis to single or small numbers of cells. Here we describe results obtained with a rapid and highly optimized global reverse transcription#150;PCR (RT-PCR) procedure. Contrary to prevalent expectations, the exponential approach preserves abundance relationships through amplification as high as 3 x 10(11)-fold. Further, it reduces by a million-fold the input amount of RNA needed for microarray analysis, and yields reproducible results from the picogram range of total RNA obtainable from single cells.
Similar articles
- [Use of the real-time RT-PCR method for investigation of small stable RNA expression level in human epidermoid carcinoma cells A431].
Nikitina TV, Nazarova NIu, Tishchenko LI, Tuohimaa P, Sedova VM. Nikitina TV, et al. Tsitologiia. 2003;45(4):392-402. Tsitologiia. 2003. PMID: 14520871 Russian. - [Reverse transcriptase PCR (RT-PCR) and quantitative-competitive PCR (QC-PCR)].
Chung HW. Chung HW. Exp Mol Med. 2001 Apr 21;33(1 Suppl):85-97. Exp Mol Med. 2001. PMID: 11708328 Korean. - An alternative method to amplify RNA without loss of signal conservation for expression analysis with a proteinase DNA microarray in the ArrayTube format.
Schüler S, Wenz I, Wiederanders B, Slickers P, Ehricht R. Schüler S, et al. BMC Genomics. 2006 Jun 12;7:144. doi: 10.1186/1471-2164-7-144. BMC Genomics. 2006. PMID: 16768788 Free PMC article. - cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.
Hillmann A, Dunne E, Kenny D. Hillmann A, et al. Methods Mol Biol. 2009;496:223-43. doi: 10.1007/978-1-59745-553-4_15. Methods Mol Biol. 2009. PMID: 18839114 Review. - Transcriptome amplification methods in gene expression profiling.
Peano C, Severgnini M, Cifola I, De Bellis G, Battaglia C. Peano C, et al. Expert Rev Mol Diagn. 2006 May;6(3):465-80. doi: 10.1586/14737159.6.3.465. Expert Rev Mol Diagn. 2006. PMID: 16706747 Review.
Cited by
- Expression of microRNA processing machinery genes in rhesus monkey oocytes and embryos of different developmental potentials.
Mtango NR, Potireddy S, Latham KE. Mtango NR, et al. Mol Reprod Dev. 2009 Mar;76(3):255-69. doi: 10.1002/mrd.20950. Mol Reprod Dev. 2009. PMID: 18646051 Free PMC article. - T7-based linear amplification of low concentration mRNA samples using beads and microfluidics for global gene expression measurements.
Kralj JG, Player A, Sedrick H, Munson MS, Petersen D, Forry SP, Meltzer P, Kawasaki E, Locascio LE. Kralj JG, et al. Lab Chip. 2009 Apr 7;9(7):917-24. doi: 10.1039/b811714d. Epub 2008 Dec 15. Lab Chip. 2009. PMID: 19294302 Free PMC article. - Single cells get together: High-resolution approaches to study the dynamics of early mouse development.
Saiz N, Plusa B, Hadjantonakis AK. Saiz N, et al. Semin Cell Dev Biol. 2015 Dec;47-48:92-100. doi: 10.1016/j.semcdb.2015.06.004. Epub 2015 Jul 13. Semin Cell Dev Biol. 2015. PMID: 26183190 Free PMC article. Review. - A sequence-based identification of the genes detected by probesets on the Affymetrix U133 plus 2.0 array.
Harbig J, Sprinkle R, Enkemann SA. Harbig J, et al. Nucleic Acids Res. 2005 Feb 18;33(3):e31. doi: 10.1093/nar/gni027. Nucleic Acids Res. 2005. PMID: 15722477 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources