mRNA amplification for microarray analysis
mRNA amplification protocols for array analysis:
Protocol for mRNA amplification and target preparation
(Written by Max Diehn (Stanford University) based on Wang et. al., Nature Biotechnology, 2000)
Protocol for mRNA amplification and target preparation (NCI maDB)
Reduced Volume RNA Amplification Protocol (Scripps institute, a modified version of that published by Baugh et al., 2001.)
Starting material and rounds of amplification:
? 1.0 ug totRNA ? 1 Round amplification
? 1.0 ng totRNA ? 2 Rounds amplification
? 1.0 pg totRNA ? 3 Rounds amplification
Troubleshooting mRNA amplification:
- RNA quality is poor.
- RNA starting amount is not enought.
- Primer concentration or purity is poor (T7 oligo-dT primer
- cDNA amplification program is wrong.
Key literatures:
Nat Biotechnol. 2000 Apr;18(4):457-9.
High-fidelity mRNA amplification for gene profiling. Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM.
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.
Nucleic Acids Res. 2001 March 1; 29(5): e29.
Quantitative analysis of mRNA amplification by in vitro transcription. L. R. Baugh, A. A. Hill, E. L. Brown, and Craig P. Hunter.
Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.
Last update 25-Sep-2006, Rating n/a of 0 votes.
