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PROVIDER: PRJNA94913 | 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: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:Effect of ligase on T7 based RNA linear amplification
| PRJNA104835 | ENA
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:Effect of column cleanup on T7 based RNA linear amplification
| PRJNA104837 | ENA
Project description:Effects of template switching (TS) primer and cDNA cleanup columns on T7 based RNA linear amplification
| PRJNA104833 | ENA
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
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:We have developed a method of mRNA amplification based on T7 polymerase, where T7 promoter is attached to a oligonucleotide containing the mini-exon sequence, which will hybridize with cDNA molecules containing the anti-ME sequence
2017-07-31 | GSE94766 | GEO