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| Status |
Public on Mar 07, 2006 |
| Title |
Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
|
| Summary |
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.
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| Overall design |
Refer to individual Series
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| Citation(s) |
12445333 |
| |
| Submission date |
Mar 06, 2006 |
| Last update date |
Dec 28, 2012 |
| Organization |
Stanford Microarray Database (SMD) |
| E-mail(s) |
array@genome.stanford.edu
|
| Phone |
650-498-6012
|
| URL |
http://genome-www5.stanford.edu/
|
| Department |
Stanford University, School of Medicine
|
| Street address |
300 Pasteur Drive
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
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| Platforms (4)
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| Samples (73)
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| This SuperSeries is composed of the following SubSeries:
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| 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 |
|
| Relations |
| BioProject |
PRJNA94913 |
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