Homo sapiens
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PROVIDER: PRJNA104833 | ENA |
REPOSITORIES: ENA
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Project description:Zhao et al. Amplification Table 1 This experiment was designed to determine the effects of template switching (TS) primer and the type of columns used in ds cDNA cleanup on the fidelity of the T7 based RNA linear amplification. BC91 total RNA was amplified with or without TS primer and with two different ds cDNA cleanup protocols. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2005-11-04 | GSE3560 | GEO
Project description:Zhao et al. Amplification Table 1 This experiment was designed to determine the effects of template switching (TS) primer and the type of columns used in ds cDNA cleanup on the fidelity of the T7 based RNA linear amplification. BC91 total RNA was amplified with or without TS primer and with two different ds cDNA cleanup protocols. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
2005-11-03 | E-GEOD-3560 | biostudies-arrayexpress
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: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: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: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:Zhao et al. Amplification Table 9 This experiment was designed to evaluate the effect of the amount of input total RNA on the fidelity, reproducibility, and yield of T7 based RNA linear amplification. BC2 total RNA was amplified using the Jeffrey lab protocol. Different amounts of T7 primer were used according to the quantity of input total RNA. Multiple amplifications were done for each quantity of input total RNA to minimize experimental variations. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2005-11-04 | GSE3557 | GEO
Project description:In our original grant we proposed to use the NR3B-null mouse model to study the role of NR3B subunit in motor neuron function. We have now successfully generated NR3B null mice. Interestingly, NR3B-null mice invariably die at age P4-P8. Our preliminary examination indicates that the motor strength of these mice is severely impaired prior to death. As we continue to explore the cause of death in NR3B null mice, we propose to conduct gene profiling experiments to search for transcription changes in the brain related to ablation of the NR3B gene. We have previously used the UCLA array facility provided by the NINDS/NIMH Microarray Consortium to identify genes that show abnormal expression patterns in these mice using whole brain samples. Now, we would like to use the same approach to analyze pure motor neuron samples obtained by Laser Capture Microdisection (LCM). As these samples are of small size we need to use RNA amplification method. We would like to compare probes from two amplification methods to see which one gives the best result. We will collect the cDNA probe obtained by RNA-based single primer isothermal amplification developed by Nugen, Inc. and the aRNA probe obtained from T7-based RNA amplification. Both the hybridization intensity and the percentage of the present call of these two types of probe will be analyzed. The cDNA probe obtained by RNA-based single primer isothermal amplification developed by Nugen, Inc. may have some advantage over the aRNA probe obtained from T7-based RNA amplification. Two samples will be processed by either RNA-based single primer isothermal amplification developed by Nugen or T7-based RNA amplification to obtain cDNA or aRNA probes, respectively. For each sample we will use 600 motor neurons obtained by LCM. Both types of probe will be labeled by biotin and hybridize to Affymetrix GeneChip Mouse Genome 430 2.0 Array. Data analysis will be performed by array facility. Keywords: dose response
2006-06-09 | GSE5036 | GEO
Project description:Zhao et al. Amplification Table 3 This experiment was designed to evaluate the effect of ligase used in the second strand cDNA synthesis on the fidelity of T7 based RNA linear amplification. BC2 total RNA was amplified with or without ligase using Affymetrix protocol. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2005-11-04 | GSE3561 | GEO