Automation of cDNA synthesis and labelling improves reproducibility.
BACKGROUND
Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads.


RESULTS
The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures.


CONCLUSIONS
We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.
Klevebring
Daniel
D
Division of Gene Technology, School of Biotechnology, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden.
Gry
Marcus
M
Lindberg
Johan
J
Eidefors
Anna
A
Lundeberg
Joakim
J
D001331:Automation / Q000379:methods*; D018076:DNA, Complementary / Q000096:biosynthesis*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008863:Microspheres; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods*
D018076:DNA, Complementary; D005456:Fluorescent Dyes