Comparison of RNA amplification techniques meeting the demands for the expression profiling of clinical cancer samples.
ABSTRACT Available ribonucleic acid (RNA) amplification methods are extensively tested for reproducibility, but only a few studies additionally deal with potential amplification bias. On targeted arrays, we evaluated three amplification protocols, which are less time consuming than the commonly used T7-RNA polymerase based in vitro transcription protocols and therefore may be more suitable for clinical use: Template-switching polymerase chain reaction (PCR), Ribo-single primer isothermal amplification and a random primer-based PCR. Additionally, a more sensitive labelling method, Dendrimer labelling, was evaluated. All methods were compared to unamplified RNA labelled at reverse transcription. From our results, we conclude that RNA amplification with template-switching PCR is highly reproducible and results in a reliable representation of the starting RNA population. We then assessed whether RNA amplification of clinical breast and thyroid cancer samples with template-switching PCR showed robust performance when altered cycle numbers or partially degraded RNA were used. Template-switching PCR proved to be a very reliable method for global RNA amplification, even when starting from partially degraded RNA down to a RNA Integrity Number of 4.3. In conclusion, template-switching PCR amplification promises to help micro-array expression profiling of limited amounts of human samples on its way to a clinical routine.
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ABSTRACT: This report describes a method for the generation of global gene expression profiles from low frequent B-cell subsets by using fluorescence-activated cell sorting and RNA amplification. However, some of the differentiating compartments involve a low number of cells and therefore it is important to optimize and validate each step in the procedure. Normal lymphoid tissues from blood, tonsils, thymus and bone marrow were immunophenotyped by the 8-colour Euroflow panel using multiparametric flow cytometry. Subsets of B-cells containing cell numbers ranging from 800 to 33,000 and with frequencies varying between 0.1 and 10 percent were sorted, subjected to mRNA purification, amplified by the NuGEN protocol and finally analysed by the Affymetrix platform. Following a step by step strategy, each step in the workflow was validated and the sorting/storage conditions optimized as described in this report. First, an analysis of four cancer cell lines on Affymetrix arrays, using either 100 ng RNA labelled with the Ambion standard protocol or 1 ng RNA amplified and labelled by the NuGEN protocol, revealed a significant correlation of gene expressions (r >= 0.9 for all). Comparison of qPCR data in samples with or without amplification for 8 genes showed that a relative difference between six cell lines was preserved (r >= 0.9). Second, a comparison of cells sorted into PrepProtect, RNAlater or directly into lysis/binding buffer showed a higher yield of purified mRNA following storage in lysis/binding buffer (p < 0.001). Third, the identity of the B-cell subsets validated by the cluster of differentiation (CD) membrane profile was highly concordant with the transcriptional gene expression (p-values <0.001). Finally, in normal bone marrow and tonsil samples, eight evaluated genes were expressed in accordance with the biology of lymphopoiesis (p-values < 0.001), which enabled the generation of a gene-specific B-cell atlas. A description of the implementation and validation of commercially available kits in the laboratory has been examined. This included steps for cell sorting, cell lysis/stabilization, RNA isolation, RNA concentration and amplification for microarray analysis. The workflow described in this report will enable the generation of microarray data from minor sorted B-cell subsets.BMC Immunology 01/2014; 15(1):3. · 2.61 Impact Factor
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ABSTRACT: Alternative concepts for a flexible radiometer system for Sun calibration were analyzed, imposing as few constraints as possible to the spacecraft system. The principal possibilities for the variation of the instantaneous field of view (FOV) were investigated in order to provide better data correlation for the wide FOV system. The influence of polarization on the system energy transfer function was calculated. Results make it possible to specify a system with higher performance concerning system calibration and geometrical data correlation. The theoretical limits of radiometric resolution due to polarization errors are given.NASA STI/Recon Technical Report N. 02/1981;
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ABSTRACT: In this study we investigated the fidelity and representativeness of two novel multiple displacement amplification (MDA) protocols leading to whole transcriptome amplification (WTA). WTA is used to amplify a limiting amount of experimental RNA, allowing its use in downstream applications. Using Phi29 and Bst DNA polymerase-based MDA, henceforth referred to as WTA-Phi and WTA-Bst respectively, we successfully amplified very low amounts of linearly concatenated cDNA originating from 10 and 100 ng of starting RNA. The average yield obtained from 10 ng was 3.5 µg and 4.7 µg for WTA-Phi and WTA-Bst respectively, while 100 ng of starting RNA yielded 7.0 µg and 12.4 µg for WTA-Phi and WTA-Bst respectively. Representational distortion of the templates, analyzed via conventional PCR, showed robust amplification of 11 different transcripts when either WTA-Phi or WTA-Bst synthesized templates were used, while some transcripts were not detected from unamplified templates. Loci representation, a measure of amplification consistency, was evaluated using TaqMan RT-qPCR amplification of five different transcripts, yielding values ranging from 96.4% to 189.3%, comparable to those obtained using genomic target based MDA systems. The two MDA protocols described in this study efficiently lead to representative WTA, using as little as 10 ng of starting RNA.Molecular Biotechnology 10/2013; · 2.26 Impact Factor