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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    ABSTRACT: The harlequin ladybird beetle Harmonia axyridis has emerged as a model species in invasion biology because of its strong resistance against pathogens and remarkable capacity to outcompete native ladybirds. The invasive success of the species may reflect its well-adapted immune system, a hypothesis we tested by analysing the transcriptome and characterizing the immune gene repertoire of untreated beetles and those challenged with bacteria and fungi. We found that most H. axyridis immunity-related genes were similar in diversity to their counterparts in the reference beetle Tribolium castaneum, but there was an unprecedented expansion among genes encoding antimicrobial peptides and proteins (AMPs). We identified more than 50 putative AMPs belonging to seven different gene families, and many of the corresponding genes were shown by quantitative real-time RT-PCR to be induced in the immune-stimulated beetles. AMPs with the highest induction ratio in the challenged beetles were shown to demonstrate broad and potent activity against Gram-negative bacteria and entomopathogenic fungi. The invasive success of H. axyridis can therefore be attributed at least in part to the greater efficiency of its immune system, particularly the expansion of AMP gene families and their induction in response to pathogens.
    Full-text · Article · Jan 2013 · Proceedings of the Royal Society B: Biological Sciences
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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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    ABSTRACT: Background The primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs). The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae. Results Using a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH) families: GH11 (xylanases), GH28 (polygalacturonases or pectinases), and GH45 (β-1,4-glucanases or cellulases). Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs) families as well as polygalacturonase-inhibiting proteins (PGIPs) were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome analyses could be partially explained by differences in transcriptional level. Conclusions Combining proteome and transcriptome sequencing analyses proved to be a powerful tool for the discovery of active PCWDEs in a non-model species. Our data represent the starting point of an in-depth functional and evolutionary characterization of PCWDE gene families in phytophagous beetles and their contribution to the adaptation of these highly successful herbivores to their host plants.
    Full-text · Article · Nov 2012 · BMC Genomics
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    • "The longer reads allow for accurate assemblies with fewer gaps, longer contigs and better assignment of repeat regions. In addition, we normalized the cDNA library prior to the sequencing analysis, as highly abundant transcripts may impede de novo identification of low-expressed or tissue specific genes [27], [28]. Pooling and normalization of the total RNA followed by GS-FLX titanium sequencing provided a comprehensively annotated ferret reference transcriptome. "
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    ABSTRACT: The ferret is commonly used as a model for studies of infectious diseases. The genomic sequence of this animal model is not yet characterized, and only a limited number of fully annotated cDNAs are currently available in GenBank. The majority of genes involved in innate or adaptive immune response are still lacking, restricting molecular genetic analysis of host response in the ferret model. To enable de novo identification of transcriptionally active ferret genes in response to infection, we performed de-novo transcriptome sequencing of animals infected with H1N1 A/California/07/2009. We also included splenocytes induced with bacterial lipopolysaccharide to allow for identification of transcripts specifically induced by gram-negative bacteria. We pooled and normalized the cDNA library in order to delimit the risk of sequencing only highly expressed genes. While normalization of the cDNA library removes the possibility of assessing expression changes between individual animals, it has been shown to increase identification of low abundant transcripts. In this study, we identified more than 19,000 partial ferret transcripts, including more than 1000 gene orthologs known to be involved in the innate and the adaptive immune response.
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