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Abstract Background The template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. However, when applied to very small RNA samples, such as those obtained from tens or hundreds of cells, this approach leads to high background and low cDNA yield due to concatamerization of the TS oligo. Results In this study, we describe the application of nucleotide isomers that form non-standard base pairs in the template switching oligo to prevent background cDNA synthesis. When such bases are added to the 5' end of the template switching (TS) oligo, they inhibit MMLV-RT from extending the cDNA beyond the TS oligo, thus increasing cDNA yield by reducing formation of concatamers of the TS oligo that are the source of significant background. Conclusions Our results demonstrate that this novel approach for cDNA synthesis has valuable utility for application of ultra-high throughput technologies, such as whole transcriptome sequencing using 454 technology, to very small biological samples comprised of tens of cells as might be obtained via approaches like laser microdissection.

Background: Ginger (Zingiber officinale) and turmeric (Curcuma longa) accumulate important pharmacologically active metabolites at high levels in their rhizomes. Despite their importance, relatively little is known regarding gene expression in the rhizomes of ginger and turmeric. Results: In order to identify rhizome-enriched genes and genes encoding specialized metabolism enzymes and pathway regulators, we evaluated an assembled collection of expressed sequence tags (ESTs) from eight different ginger and turmeric tissues. Comparisons to publicly available sorghum rhizome ESTs revealed a total of 777 gene transcripts expressed in ginger/turmeric and sorghum rhizomes but apparently absent from other tissues. The list of rhizome-specific transcripts was enriched for genes associated with regulation of tissue growth, development, and transcription. In particular, transcripts for ethylene response factors and AUX/IAA proteins appeared to accumulate in patterns mirroring results from previous studies regarding rhizome growth responses to exogenous applications of auxin and ethylene. Thus, these genes may play important roles in defining rhizome growth and development. Additional associations were made for ginger and turmeric rhizome-enriched MADS box transcription factors, their putative rhizome-enriched homologs in sorghum, and rhizomatous QTLs in rice. Additionally, analysis of both primary and specialized metabolism genes indicates that ginger and turmeric rhizomes are primarily devoted to the utilization of leaf supplied sucrose for the production and/or storage of specialized metabolites associated with the phenylpropanoid pathway and putative type III polyketide synthase gene products. This finding reinforces earlier hypotheses predicting roles of this enzyme class in the production of curcuminoids and gingerols. Conclusion: A significant set of genes were found to be exclusively or preferentially expressed in the rhizome of ginger and turmeric. Specific transcription factors and other regulatory genes were found that were common to the two species and that are excellent candidates for involvement in rhizome growth, differentiation and development. Large classes of enzymes involved in specialized metabolism were also found to have apparent tissue-specific expression, suggesting that gene expression itself may play an important role in regulating metabolite production in these plants. [ABSTRACT FROM AUTHOR]

Background: The template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. However, when applied to very small RNA samples, such as those obtained from tens or hundreds of cells, this approach leads to high background and low cDNA yield due to concatamerization of the TS oligo. Results: In this study, we describe the application of nucleotide isomers that form non-standard base pairs in the template switching oligo to prevent background cDNA synthesis. When such bases are added to the 5' end of the template switching (TS) oligo, they inhibit MMLV-RT from extending the cDNA beyond the TS oligo, thus increasing cDNA yield by reducing formation of concatamers of the TS oligo that are the source of significant background. Conclusions: Our results demonstrate that this novel approach for cDNA synthesis has valuable utility for application of ultra-high throughput technologies, such as whole transcriptome sequencing using 454 technology, to very small biological samples comprised of tens of cells as might be obtained via approaches like laser microdissection. [ABSTRACT FROM AUTHOR]

The glandular trichome is an excellent model system for investigating plant metabolic processes and their regulation within a single cell type. We utilized a proteomics-based approach with isolated trichomes of four different sweet basil ( Ocimum basilicum L.) lines possessing very different metabolite profiles to clarify the regulation of metabolism in this single cell type. Significant differences in the distribution and accumulation of the 881 highly abundant and non-redundant protein entries demonstrated that although the proteomes of the glandular trichomes of the four basil lines shared many similarities they were also each quite distinct. Correspondence between proteomic, expressed sequence tag, and metabolic profiling data demonstrated that differential gene expression at major metabolic branch points appears to be responsible for controlling the overall production of phenylpropanoid versus terpenoid constituents in the glandular trichomes of the different basil lines. In contrast, post-transcriptional and post-translational regulation of some enzymes appears to contribute significantly to the chemical diversity observed within compound classes for the different basil lines. Differential phosphorylation of enzymes in the 2- C-methyl-d-erythritol 4-phosphate (MEP)/terpenoid and shikimate/phenylpropanoid pathways appears to play an important role in regulating metabolism in this single cell type. Additionally, precursors for different classes of terpenoids, including mono- and sesquiterpenoids, appear to be almost exclusively supplied by the MEP pathway, and not the mevalonate pathway, in basil glandular trichomes. [ABSTRACT FROM AUTHOR]