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      Reliability of T7-Based mRNA Linear Amplification Validated by Gene Expression Analysis of Human Kidney Cells Using cDNA Microarrays

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          Genome wide gene expression analysis by cDNA microarrays is often limited by minute amounts of starting RNA. We therefore tested an optimized linear RNA amplification protocol using the RiboAmp<sup>®</sup> amplification kit in the setting of cDNA microarrays. We isolated mRNA from a human kidney cell line (HK-2; ATCC) and from Universal Human Reference RNA (STR; Stratagene). After performing one and two rounds of linear RNA amplification, respectively, the amplified RNAs were co-hybridized to cDNA microarrays. Linearity and reproducibility of the individual experiments were then assessed by calculating the Pearson correlation. The intra-amplification consistency showed a correlation of 0.968 for the first round, 0.907 for the second round and 0.912 for two successive rounds of amplification. If the first round was compared to unamplified material, r was 0.925. The second round amplification yielded a correlation of 0.897 if compared to unamplified mRNA. Two rounds of amplification starting from 200 pg of mRNA compared to unamplified material resulted in a correlation of 0.868. These results indicate that linear amplification using RiboAmp<sup>®</sup> kit yields amplified RNA with a high degree of linearity and reproducibility.

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          High-fidelity mRNA amplification for gene profiling.

          The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.
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            CELL SAMPLING: Laser Capture Microdissection: Molecular Analysis of Tissue

             R Bonner (1997)

              Author and article information

              Nephron Exp Nephrol
              Cardiorenal Medicine
              S. Karger AG
              July 2004
              17 November 2004
              : 97
              : 3
              : e86-e95
              aDepartment of Nephrology, University Hospital of Innsbruck, Innsbruck, bEmergentec Business Analytics, Vienna, and cTyrolean Cancer Research Institute, Innsbruck, Austria; dDepartment of Nephrology, Stanford University Medical School, Stanford, Calif., USA
              78642 Nephron Exp Nephrol 2004;97:e86–e95
              © 2004 S. Karger AG, Basel

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              Page count
              Figures: 2, Tables: 4, References: 13, Pages: 1
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              Original Paper

              Cardiovascular Medicine, Nephrology

              RNA, Gene expression, Microarrays, Kidney, Amplification


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