title: Fidelity and representativeness of two isothermal multiple displacement amplification systems to preamplify limiting amounts of total RNA.
abstract: In this study we investigated the fidelity and representativeness of two novel multiple displacement amplification (MDA) protocols leading to whole transcriptome amplification (WTA). WTA is used to amplify a limiting amount of experimental RNA, allowing its use in downstream applications. Using Phi29 and Bst DNA polymerase-based MDA, henceforth referred to as WTA-Phi and WTA-Bst, respectively, we successfully amplified very low amounts of linearly concatenated cDNA originating from 10 to 100 ng of starting RNA. The average yield obtained from 10 ng was 3.5 and 4.7 μg for WTA-Phi and WTA-Bst, respectively, while 100 ng of starting RNA yielded 7.0 and 12.4 μg for WTA-Phi and WTA-Bst, respectively. Representational distortion of the templates, analyzed via conventional PCR, showed robust amplification of 11 different transcripts when either WTA-Phi or WTA-Bst synthesized templates were used, while some transcripts were not detected from unamplified templates. Loci representation, a measure of amplification consistency, was evaluated using TaqMan RT-qPCR amplification of five different transcripts, yielding values ranging from 96.4 to 189.3 %, comparable to those obtained using genomic target-based MDA systems. The two MDA protocols described in this study efficiently lead to representative WTA, using as little as 10 ng of starting RNA.
authors.affiliation: Department of Biology, Université de Moncton, 18 Antonine-Maillet Ave, Moncton, NB, E1A 3E9, Canada.
authors.forename: Vijay J
authors.initials: VJ
authors.lastname: Gadkar
authors.forename: Tanya
authors.initials: T
authors.lastname: Arseneault
authors.forename: Martin
authors.initials: M
authors.lastname: Filion
mesh_terms: D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D018745:Genome, Plant; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D011198:Solanum tuberosum / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D012313:RNA; D004259:DNA-Directed DNA Polymerase