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High-fidelity mRNA amplification for gene profiling
E Wang1, L D Miller, G A Ohnmacht
1Surgery Branch, Division of Clinical Sciences, National Cancer Institute and the Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA.
Abstract:
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.
Insights
This study introduces a novel RNA amplification method for cDNA microarrays, enabling gene expression analysis with significantly less starting material. The optimized procedure maintains high fidelity, expanding microarray applications in research and diagnostics.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- The Human Genome Project facilitates comprehensive gene expression analysis.
- cDNA microarrays are used for expression analysis in cancer research.
- Limited RNA quantity restricts broader cDNA microarray application.
Purpose of the Study:
- To develop an optimized RNA amplification procedure for low-abundance samples.
- To assess the limits and biases of RNA amplification methods for cDNA microarrays.
- To enable broader application of cDNA microarrays by reducing RNA input requirements.
Main Methods:
- Combined antisense RNA (aRNA) amplification with a template-switching effect.
- Optimized amplification for low-abundance RNA samples.
- Assessed fidelity of amplified RNA using cDNA microarrays.
Main Results:
- The devised procedure effectively amplifies low-abundance RNA samples.
- Amplified RNA fidelity was comparable to conventional methods using poly(A) RNA or total RNA.
- Expression profiles remained consistent across various input RNA dilutions (1:10,000 to 1:100,000).
Conclusions:
- The novel amplification method significantly reduces the RNA input needed for cDNA microarrays.
- This technique enhances the utility of cDNA microarrays for gene expression profiling.
- The optimized procedure offers a reliable approach for analyzing limited RNA samples in research.