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Sequence-independent Selective Amplification of mRNAs over rRNAs
EP20382
Poster Title: Sequence-independent Selective Amplification of mRNAs over rRNAs
Submitted on 19 Dec 2013
Author(s): John Arrand1, Sim Sihota1, Wenbin Wei1 and Guido Krupp2
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Poster Views: 1,427
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Poster Information
Abstract: Standard mRNA amplifications for "All-Exon" microarrays and for bacterial RNAs are impossible with small samples (<100 ng total RNA) and with degraded RNAs, because removal of rRNAs must precede universal, non-selective RNA amplification (Affymetrix and Ambion kits, respectively). This pre-treatment with magnetic beads is cumbersome, requires high amounts of starting material (>0.5 mg total RNA), is not universal for all species and degraded RNAs are not suitable. Omission of this step results in lost sensitivity.

However, AmpTec's unique TRinucleotide primer strategy renders reverse transcriptions selective for mRNA (fragments) and it provides sequence independent,preferential 3'-proximal priming. This eliminates the rRNA removal step, thus high mRNA amplifications (input of <1 ng total RNA yields >10 mg amplified RNAs), and the use of degraded RNAs are possible.

A first Exon Array study with saliva RNA (<4ng of degraded RNA) is now in press [2].

[1] Pepper et al., 2007 “A Core Lab Case Study: Exon Array Challenges and Opportunities” Affymetrix Application Note.

[2] Hu et al., 2008 (Wong lab at UCLA) “Exon-level expression profiling: a comprehensive transcriptome analysis for oral fluids.”
Clinical Chemistry 54:5 (E-publication ahead of print).
Summary: Standard mRNA amplifications for "All-Exon" microarrays and for bacterial RNAs are impossible with small samples and with degraded RNAs. This is because the removal of rRNAs must precede universal, non-selective RNA amplification. This pre-treatment with magnetic beads is cumbersome, requires high amounts of starting material and is not universal for all species as well as the fact that degraded RNAs are not suitable.
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