Accessing the Transcriptome: How to Normalize mRNA Pools

Department of Entomology, Max Planck Institute for Chemical Ecology, Jena, Germany.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2011; 772:105-28. DOI: 10.1007/978-1-61779-228-1_6
Source: PubMed


As advances in next generation sequencing continue to provide increasing access to the genomics -revolution for research systems having few or no genomic resources, transcriptome sequencing will only increase in importance as a fast and direct means of accessing the genes themselves. However, constructing a comprehensive cDNA library for deep sequencing is very difficult, as highly abundant transcripts hamper de novo identification of low-expressed genes, and genes expressed only under very specific conditions will remain elusive. The reduction of variance in gene expression levels to within a tenfold range of differences by cDNA normalization provides an important means of allocating sequencing across a greater fraction of genes, directly translating into a more even coverage across genes. Here, we outline two different normalization methods, addressing many of the important issues we think need consideration when going from RNA isolation to the cDNA material required for sequencing. This will provide coding gene information across thousands of genes from any organism, providing rapid insights into topics such as gene family member identification and genetic variation that may be associated with a studied phenotype.

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Available from: Heiko Vogel, Apr 08, 2014
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    • "Normalization was achieved by one cycle of denaturation and reassociation, resulting in a mixture of single-stranded and double-stranded cDNAs. Reassociated double-stranded cDNA was separated from the normalized cDNA by passing the mixture over a hydroxyapatite column and amplifying the single cDNA strands for 11 cycles [17]. The resulting double-stranded cDNA pool was purified and concentrated using the DNA Clean and Concentrator Kit (Zymogen) and size fractionated on SizeSep 400 spun columns (GE Healthcare) with a cutoff of approximately 150 bp. "
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    • "The procedure generally followed the manufacturer’s protocol but included several important modifications,as described [57]. Optimization of the complete cDNA normalization procedure was performed as described in [58]. The resulting normalized cDNA library was used for 454 pyrosequencing using the Roche 454 FLX machine and Sanger sequencing on an ABI 3730 xl automatic DNA sequencer (PE Applied Biosystems). "
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