Sequence-independent Selective Amplification of mRNAs over rRNAs
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 µg total RNA), is not universal for all species
and degraded RNAs are not suitable. Omission of this step results in lost sensitivity [1].
However, AmpTec's unique TRinucleotide primer strategy renders reverse transcriptions selective for mRNA (fragments) and it provides sequenceindependent, preferential 3'-proximal priming. This eliminates the rRNA removal step, thus high mRNA amplifications (input of <1 ng total RNA
yields >10 µg 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).
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 µg total RNA), is not universal for all species
and degraded RNAs are not suitable. Omission of this step results in lost sensitivity [1].
However, AmpTec's unique TRinucleotide primer strategy renders reverse transcriptions selective for mRNA (fragments) and it provides sequenceindependent, preferential 3'-proximal priming. This eliminates the rRNA removal step, thus high mRNA amplifications (input of <1 ng total RNA
yields >10 µg 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).
