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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

New Agilent platform DNA microarrays for transcriptome analysis of Plasmodium falciparum and Plasmodium berghei for the malaria research community

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Björn F C Kafsack, Heather J Painter, Manuel Llinás
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-187) contains supplementary material, which is available to authorized users.
Björn F C Kafsack, Heather J Painter contributed equally to this work.

Competing interests

The authors declare they have no conflicts of interest.

Authors' contributions

BK designed the arrays, analysed the data, and contributed equally to the generation of figures and tables. HJP developed the experimental protocols, performed the experiments and contributed equally to the generation of figures and tables. ML contributed to the design of the microarrays and analysis. All authors contributed to experimental design and to writing the manuscript. All authors read and approved the final manuscript.

Abstract

Background

DNA microarrays have been a valuable tool in malaria research for over a decade but remain in limited use in part due their relatively high cost, poor availability, and technical difficulty. With the aim of alleviating some of these factors next-generation DNA microarrays for genome-wide transcriptome analysis for both Plasmodium falciparum and Plasmodium berghei using the Agilent 8x15K platform were designed.

Methods

Probe design was adapted from previously published methods and based on the most current transcript predictions available at the time for P. falciparum or P. berghei. Array performance and transcriptome analysis was determined using dye-coupled, aminoallyl-labelled cDNA and streamlined methods for hybridization, washing, and array analysis were developed.

Results

The new array design marks a notable improvement in the number of transcripts covered and average number of probes per transcript. Array performance was excellent across a wide range of transcript abundance, with low inter-array and inter-probe variability for relative abundance measurements and it recapitulated previously observed transcriptional patterns. Additionally, improvements in sensitivity permitted a 20-fold reduction in necessary starting RNA amounts, further reducing experimental costs and widening the range of application.

Conclusions

DNA microarrays utilizing the Agilent 8x15K platform for genome-wide transcript analysis in P. falciparum and P. berghei mark an improvement in coverage and sensitivity, increased availability to the research community, and simplification of the experimental methods.
Zusatzmaterial
Additional file 1: Excel file of P. falciparum v7.1 8x15K microarray probes. (XLSX 1003 KB)
12936_2012_2126_MOESM1_ESM.xlsx
Additional file 2: Excel file of P. berghei Mar2011 8x15K microarray probes. (XLSX 934 KB)
12936_2012_2126_MOESM2_ESM.xlsx
Additional file 3: Figure illustrating coverage for three arrays. (PDF 131 KB)
12936_2012_2126_MOESM3_ESM.pdf
Additional file 4: Figures and table describing array-wide technical reproducibility of hybridizations across three replicates. (PDF 751 KB)
12936_2012_2126_MOESM4_ESM.pdf
Additional file 5: Table of titration series of total RNA starting amounts, resultant cDNA generation, dye-coupling, and array hybridization. (PDF 63 KB)
12936_2012_2126_MOESM5_ESM.pdf
Additional file 6: Table of transcripts well-above background by amount hybridized and table of signal intensity correlation across a self-hybridization dilution series. (PDF 89 KB)
12936_2012_2126_MOESM6_ESM.pdf
Additional file 7: List of highly expressed periodic genes used in Figure 3. (XLSX 35 KB)
12936_2012_2126_MOESM7_ESM.xlsx
Authors’ original file for figure 1
12936_2012_2126_MOESM8_ESM.jpeg
Authors’ original file for figure 2
12936_2012_2126_MOESM9_ESM.pdf
Authors’ original file for figure 3
12936_2012_2126_MOESM10_ESM.jpeg
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