Comparison of genomic DNA amplification methods
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE8449 | GEO | 2008/11/01
SECONDARY ACCESSION(S): PRJNA101525
REPOSITORIES: GEO
Similar Datasets
Project description:Hybridization of amplified genomic DNA against unamplified genomic DNA using three different amplification methods to assess the bias introduced by amplification alone. Keywords: genomic DNA Three different amplification methods were assessed and total genomic DNA hybridized against equal amounts of amplified genomic DNA for each method.
2010-05-17 | E-GEOD-8449 | biostudies-arrayexpress
Project description:Background The amplification of bacterial RNA is required if complex host-pathogen interactions are to be studied where the recovery of bacterial RNA is limited. Here, using a whole genome Mycobacterium tuberculosis microarray to measure cross-genome representation of amplified mRNA populations, we have investigated two approaches to RNA amplification using different priming strategies. The first using oligo-dT primers after polyadenylation of the bacterial RNA, the second using a set of mycobacterial amplification-directed primers. Results The reproducibility, sensitivity, and the representational bias introduced by these amplification systems were examined by contrasting expression profiles of the amplified products from inputs of 500, 50 and 5ng total M.tuberculosis RNA with unamplified RNA from the same source. In addition a model M.tuberculosis system of microaerophilic growth and non-replicating persistence was used to assess the capability of amplified RNA microarray comparisons from an experimental system where RNA yield is restricted. Mycobacterial RNA was reproducibly amplified using both methods from as little as 5ng total RNA (~ equivalent to 2E+05 bacilli). Differential gene expression patterns observed with unamplified RNA in the switch from aerobic to microaerophilic growth were also reflected in the amplified expression profiles. Conclusions Here we describe two reproducible methods of bacterial RNA amplification that will allow previously intractable host-pathogen interactions during bacterial infection to be explored at the whole genome level by RNA profiling. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-42
2008-03-12 | E-BUGS-42 | biostudies-arrayexpress
Project description:We tested the performance of three methods for amplifying single-cell amounts of RNA under ideal conditions: T7-based in vitro transcription; switching mechanism at 5' end of RNA template (SMART) PCR amplification; and global PCR amplification. All methods introduced amplification-dependent noise when mRNA was amplified 108-fold, compared with data from unamplified cDNA. PCR-amplified cDNA demonstrated the smallest number of differences between two parallel replicate samples and the best correlation between independent amplifications from the same cell type, with SMART outperforming global PCR amplification. SMART had the highest true-positive rate and the lowest false-positive rate when comparing expression between two different cell types, but had the lowest absolute discovery rate of all three methods. Direct comparison of the performance of SMART and global PCR amplification on single-cell amounts of total RNA and on single neural stem cells confirmed these findings. Under the conditions tested, PCR amplification was more reliable than linear amplification for detecting true expression differences between samples. SMART amplification had a higher true-positive rate than global amplification, but at the expense of a considerably lower absolute discovery rate and a systematic compression of observed expression ratios. Keywords: Oliginucleotide expression microarrays, T7-based linear amplification; SMART PCR-based amplification; global PCR amplification
2006-06-27 | GSE5136 | GEO
Project description:Abstract: BACKGROUND: T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. RESULTS: Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. CONCLUSION: T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. This SuperSeries is composed of the SubSeries listed below.
2006-03-07 | GSE4349 | GEO
Project description:Considerable variation in gene expression data from different DNA microarray platforms has been demonstrated. However, no characterization of the source of variation arising from labeling protocols has been performed. To analyze the variation associated with T7-based RNA amplification/labeling methods, aliquots of the Stratagene Human Universal Reference RNA were labeled using 3 eukaryotic target preparation methods and hybridized to a single array type (Affymetrix U95Av2). Variability was measured in yield and size distribution of labeled products, as well as in the gene expression results. All methods showed a shift in cRNA size distribution, when compared to un-amplified mRNA, with a significant increase in short transcripts for methods with long IVT reactions. Intra-method reproducibility showed correlation coefficients >0.99, while inter-method comparisons showed coefficients ranging from 0.94 to 0.98 and a nearly two-fold increase in coefficient of variation. Fold amplification for each method was positively correlated with the number of present genes. Two factors that introduced significant bias in gene expression data were observed: a) number of labeled nucleotides that introduces sequence dependent bias, and b) the length of the IVT reaction that introduces a transcript size dependent bias. This study provides evidence of amplification method dependent biases in gene expression data. Keywords: method validation study
2006-01-01 | GSE3254 | GEO
Project description:Abstract: Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalin-fixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. This SuperSeries is composed of the SubSeries listed below.
2006-03-15 | GSE4467 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE3557: Effect of the amount of input total RNA on T7 amplification GSE3558: Effect of in vitro transcription time on the fidelity of T7-based RNA linear amplification GSE3559: Variation in cDNA microarray analysis of gene expression using unamplified poly(A)+ RNA GSE3560: Effects of template switching (TS) primer and cDNA cleanup columns on T7 based RNA linear amplification GSE3561: Effect of ligase on T7 based RNA linear amplification GSE3562: Effect of column cleanup on T7 based RNA linear amplification GSE3563: Correlation between expression levels of different tumors measured by poly(A)+RNA and aRNA Abstract: BACKGROUND: T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. RESULTS: Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. CONCLUSION: T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. Refer to individual Series
2006-03-06 | E-GEOD-4349 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE4078: Phi29-based amplification of fresh-frozen and FFPE tumor DNA GSE4079: Genome-Wide Gene Dosage Representation in Phi29-Amplified DNA Abstract: Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalin-fixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. Refer to individual Series
2006-03-14 | E-GEOD-4467 | biostudies-arrayexpress
Project description:For microarray experiments starting with nanogram amounts of RNA it is essential to implement reproducible and powerful RNA amplification techniques. Available methods were mainly tested for reproducibility, only a few studies concentrated on potential amplification bias. 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 (TS)-PCR (SMART-PCR Kit, BD), Ribo-SPIA (single primer isothermal amplification, Oviation, Nugen) and a random primer-based PCR. Additionally a more sensitive labeling method, Dendrimer-labeling (Genisphere), was evaluated. All methods were compared to unamplified RNA labelled at reverse transcription. Hybridizations were carried out on a targeted two-colour oligonucleotide microarray. From our results we conclude that RNA amplification with TS-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 TS-PCR showed robust performance when altered cycle numbers or partially degraded RNA were used. According to our experiments TS-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 (RIN) of 4.3. Keywords: microarray expression profiling, RNA amplification techniques, RNA integrity
2007-08-24 | GSE5588 | GEO
Project description:Single-cell human genome analysis using whole-genome amplified product is hampered by allele bias during amplification. Using an oligonucleotide SNP array, we examined the nature of the allele bias and its effect on the chromosomal copy number analysis. Experiment Overall Design: TB106, lymphoblastoid cell line; CMK11-5 and CMK86, cell line; WGA, whole-genome amplified from bulk DNA; SC, whole-genome amplified product from single cell. Genotype and copy number status were compared between non-amplified products and their single cell products.
2008-06-16 | E-GEOD-8567 | biostudies-arrayexpress