Related Experiment Videos
A comparative analysis of data generated using two different target preparation methods for hybridization to
David Gold1, Kevin Coombes, Dina Medhane
1Department of Biostatistics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA. dgold@odin.mdacc.tmc.edu
BMC Genomics
|January 8, 2004
Summary
The double in vitro transcription (dIVT) protocol effectively identifies differentially expressed genes using nanogram quantities of total RNA. This method is highly desirable for biological systems with limited sample amounts, despite a slight loss in sensitivity.
Area of Science:
- Molecular Biology
- Genomics
- Transcriptomics
Background:
- Isolating homogenous cell populations for transcript profiling often yields limited RNA.
- RNA amplification methods are necessary but require careful evaluation for expression profile distortion.
Purpose of the Study:
- To compare the efficacy of in vitro transcription (IVT) and double in vitro transcription (dIVT) protocols for gene expression profiling.
- To evaluate RNA amplification methods for transcript profiling using limited RNA samples.
Main Methods:
- High-density oligonucleotide microarrays were used to compare IVT (5 µg total RNA) and dIVT (200 ng total RNA) protocols.
- Differential gene expression analysis was performed using a statistical model and hierarchical clustering.
- Quantitative real-time PCR was used for validation of selected genes.
Main Results:
- Both IVT and dIVT protocols detected differentially expressed genes and functional categories.
- The dIVT protocol detected approximately 10% fewer genes compared to IVT.
- Biological variation between samples exceeded protocol-introduced variation, with dIVT showing high concordance with IVT results.
Conclusions:
- The dIVT protocol enables identification of differentially expressed genes and functional categories using nanogram amounts of total RNA.
- A minor loss in sensitivity (~10%) was observed with dIVT, but its low RNA requirement makes it suitable for limited sample scenarios.
- The dIVT protocol is a valuable tool for transcript profiling in biological systems with restricted sample availability.