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Whole-transcriptome evaluation by next-generation sequencing (NGS) has been widely applied in the investigation of diverse transcriptional scenarios. In many clinical situations, including needle biopsy samples or laser microdissected cells, limited amounts of RNA are usually available for the assessment of the whole transcriptome. Here, we describe an mRNA amplification protocol based on in vitro T7 transcription for transcriptome evaluation by NGS. Initially, we performed RNAseq from two human mammary epithelial cell lines and evaluated several aspects of the transcriptomes generated by linear amplification of Poly (A)+ mRNA species, including transcript representation, variability and abundance. Our protocol showed to be efficient with respect to full-length transcript coverage and quantitative expression levels. We then evaluated the applicability of using this protocol in a more realistic research scenario, analyzing tumor tissue samples microdissected by laser capture. In order to increase the quantification power of the libraries only the 3′ end of transcripts were sequenced. We found highly reproducible RNAseq data among amplified tumor samples, with a median Spearman's correlation of 80%, strongly suggesting that the amplification step and library protocol preparation lead to a consistent transcriptional profile. Altogether, we established a robust protocol for assessing the polyadenylated transcriptome derived from limited amounts of total RNA that is applicable to all NGS platforms.

Linear-after-the-exponential (LATE)-PCR describes a novel approach to asymmetric PCR which uses adjusted melting temperatures of the limited primer to increase PCR efficiency. In this proof-of-principle study we show that linear amplification is possible over a wide range of amplification cycles. The curve characteristics of the real time PCR show that the plateau effect of conventional PCR can be avoided by this innovative technique.

Representational Difference Analysis (RDA) is a powerful but also quite complex and difficult method to reveal the variable genes among highly similar genomes or transcriptomes. This report is on the substantial simplification of RDA for microdiversity studies on Bacillus isolates. Following improvements are demonstrated: the average fragment size has been pushed up ( approximately 700 bp) by using nucleotide-biased restriction enzymes, in this case the "high-GC" enzyme HhaI for low GC Gram-positive Bacillus. A PCR-based production of the DRIVER has been applied to avoid subculturing of strains and repeated large-scale DNA extraction. Tedious estimation and adjustment of DNA concentrations for the subtractive hybridization step were by-passed by coupling DRIVER and TESTER production. The problem of linear amplification of TESTER/DRIVER hybrids has been eliminated by applying two different adapters, one for the TESTER but also another one for the DRIVER. Hence, only one RDA round is required to achieve clear bands without background. After doing this streamlined DRIVER and TESTER production, the RDA analysis may now be performed in one night and day. This simplification will allow to scale up the number of genomes under investigation.

A minor groove binder (MGB) TaqMan real-time PCR assay was developed for the detection of respiratory syncytial virus (RSV) in clinical specimens. Upon evaluation of the assay, notable differences were observed in the overall fluorescent response obtained from RSV positive specimens, with some linear amplification curves deviating only slightly from baseline fluorescence. Sequencing of the probes targets in these RSV strains revealed single base mismatches with the MGB TaqMan probe. Overall, these results highlight the usefulness of MGB TaqMan probes for the detection of mismatches, but suggest that MGB Taqman probes have limitations for routine screening for uncharacterised viral strains.

The incorporation of amorphous semiconductor devices in several circuit applications is presented considering such aspects as linear amplification, ternary threshold logic, and clocked binary gates. The circuit descriptions are brief in favor of presenting the principles of operation. Detail design data are available from laboratory reports and published papers appropriately cited in the paper. Working laboratory models were “breadboarded” for each of the circuits, however, it is cautioned that a hand selection of devices was required. Furthermore, no attempt in optimization was made. It is believed that with modification, while retaining basic principles considerable improvement in circuit performance can be achieved. It may be that crystalline semiconductor devices would avail circuits usually simpler in concept and design than those which are described here. However, the virtues of the circuits discussed herein, lie in their novelty as an application of new circuit elements that may yield ideas for improving their methods of application or give insight into new methods of employing these devices.