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A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations
Published on: October 25, 2018
Big results from small samples: evaluation of amplification protocols for gene expression profiling.
Agnes Viale1, Juan Li, Jay Tiesman
1Bionomics Research and Technology Center, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08857, USA.
Journal of Biomolecular Techniques : JBT
|June 28, 2007
Summary
New RNA amplification chemistries enable microarray analysis from minimal biological samples. Evaluation revealed significant differences in sensitivity and accuracy between methods, impacting gene expression results.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Microarrays allow quantification of thousands of transcripts but require substantial biological material.
- Limited sample availability restricts microarray applications in many biological research areas.
Purpose of the Study:
- To evaluate four RNA amplification chemistries designed for low-input samples (nanogram total RNA).
- To assess the reproducibility, sensitivity, accuracy, and comparability of these chemistries against standard T7 amplification.
Main Methods:
- Evaluation of four RNA amplification chemistries using nanogram amounts of total RNA.
- Assessment of reproducibility, sensitivity, and accuracy.
- Comparison with results from a single round of T7 amplification.
- Polymerase Chain Reaction (PCR) validation to analyze probe sequence interactions.
Main Results:
- No false-positive differential expression measurements were observed across the tested chemistries.
- Significant variations in sensitivity and accuracy were detected among the evaluated RNA amplification chemistries.
- PCR validation demonstrated that probe sequence on the array interacts with target labeling chemistry, causing chemistry-dependent probe set sensitivity that varied by over an order of magnitude.
Conclusions:
- RNA amplification chemistries for low-input samples exhibit variable performance in terms of sensitivity and accuracy.
- The choice of amplification chemistry significantly influences gene expression profiling results, particularly due to probe-dependent interactions.
- Further optimization is needed to standardize low-input RNA amplification for reliable microarray analysis.
