Use of an Isothermal Linear Amplification Method with Small Samples on DNA Microarrays
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PROVIDER: PRJNA91355 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Experiment 1: U133A arrays (2) hybridized to duplicate sscDNA samples prepared from 20 ng Clontech UHR RNA Experiment 2: Mu6500A arrays (6) hybridized to triplicate sscDNA samples prepared from 3 x 5 ng mouse liver RNA and 3 x 100 ng mouse liver RNA Experiment 3: U95Av2 arrays (6) hybridized to triplicate sscDNA samples prepared from 3 x 10 ng K562 RNA and 3 x 10 ng Stratagene UHR RNA Experiment 4: U95Av2 array (1) hybridized to sscDNA sample prepared from template minus reaction (negative control) Keywords: parallel sample
2005-05-25 | GSE2252 | GEO
Project description:Experiment 1: U133A arrays (2) hybridized to duplicate sscDNA samples prepared from 20 ng Clontech UHR RNA Experiment 2: Mu6500A arrays (6) hybridized to triplicate sscDNA samples prepared from 3 x 5 ng mouse liver RNA and 3 x 100 ng mouse liver RNA Experiment 3: U95Av2 arrays (6) hybridized to triplicate sscDNA samples prepared from 3 x 10 ng K562 RNA and 3 x 10 ng Stratagene UHR RNA Experiment 4: U95Av2 array (1) hybridized to sscDNA sample prepared from template minus reaction (negative control) Keywords: parallel sample
2005-05-25 | E-GEOD-2252 | biostudies-arrayexpress
Project description:DNA methylation plays a crucial role in development and disease. Bisulfite-free methods such as enzymatic methyl sequencing (EM-seq) offer gentle approaches for whole-genome analysis, but they typically depend on PCR-based library amplification, which distorts coverage and methylation quantification. Here, we introduce FINE (Fast Isothermal amplification via Nicking Enzyme), a robust isothermal amplification strategy that leverages a nicking enzyme-assisted strand displacement reaction and can amplify methylation libraries from sub-nanogram inputs in 20 minutes. FINE-EM-seq increases library efficiency and coverage uniformity compared to PCR-based approaches, minimizing amplification bias and improving methylation calling accuracy. Applied to zebrafish embryos at four early developmental stages, FINE-EM-seq characterized stage-associated methylation dynamics through blastulation and early gastrulation. Furthermore, our analysis revealed blastulation-associated differentially methylated regions (DMRs) overlapping with AT-rich regions that were previously under-covered. These results illustrate that FINE-EM-seq is a rapid, robust solution for low-input whole-genome methylation sequencing with broad utility in developmental biology and clinical epigenomics.
2026-04-09 | GSE307505 | GEO
Project description:To generate expression profiles from single embryos, a protocol for linear amplification of RNA was developed on the basis of Baugh et al. (15). Linear amplification enabled production of sufficient amounts of aRNA for the microarray experiments and has been shown to reliably amplify the initial mRNA population (15, 18-20). Approximately 5 ng of total RNA can be isolated from a blastocyst-stage embryo (21); therefore, it was necessary to carry out multiple rounds of amplification. To test the reproducibility and fidelity of the amplification procedure, a validation experiment was conducted using RNA from the kidney tissue of a newborn calf. Two replicates of the bovine kidney RNA were amplified and hybridized to the microarray. Comparisons were made between aRNA (rounds 1, 2, and 3) and unamplified RNA. Keywords: cDNA microarray
2005-11-30 | GSE3569 | 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: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 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:FINE-EM-seq: a rapid isothermal amplification method enabling comprehensive methylome profiling of zebrafish early embryos
| PRJNA1321894 | ENA
Project description:Zhao et al. Amplification Table 4 This experiment was designed to evaluate the effect of column cleanup on the fidelity and yield of T7 based RNA linear amplification. BC2 total RNA was amplified using the Jeffrey lab protocol with or without G50 cleanup. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2005-11-04 | GSE3562 | 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