Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis - PubMed (original) (raw)
Comparative Study
Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis
Veronica Luzzi et al. J Mol Diagn. 2003 Feb.
Abstract
Gene expression profiling using high density oligonucleotide arrays is a powerful method to generate an unbiased survey of a cell's transcriptional landscape. Increasingly complex biological questions require that this approach be applicable to the small numbers of cells that are obtained from sources such as laser capture microdissection (LCM) of solid tissues. In this report, we demonstrate that two rounds of transcript amplification can generate accurate and reproducible gene expression profiles using high density oligonucleotide microarrays, starting with as little as 10 ng of total RNA. Biased amplification of the 3' end of transcripts does not have a major impact on the overall transcript profile due to the 3' bias of probe sets incorporated in the array design. Furthermore, greater than 95% of all genes detected demonstrate less than a twofold difference in expression when independent tissue dissections of identical cell populations are compared. The accuracy and technical reproducibility of the method suggests that expression profiling using transcript amplification and high density oligonucleotide microarrays can be used on a routine basis.
Figures
Figure 1.
Electrophoretic analysis of aRNA targets. One microliter of each cRNA target was analyzed on an RNA LabChip and Agilent bioanalyzer. Lane 1: Molecular weight ladder with marker sizes indicated in base pairs. Lane 2: Input breast cancer total cellular RNA used for target generation. Lanes 3 and 4: Duplicate targets synthesized using “Standard protocol (A)” and starting with 10 μg of total RNA. Lanes 5 and 6: Duplicate cRNA samples generated from 10 ng of total RNA after the first round of amplification using the “RiboAmp Two-Round (D)” protocol. Lanes 7 and 8: Duplicate aRNA samples generated from 10 ng of total RNA after the second round of amplification using the “RiboAmp Two-Round (D)” protocol. To simultaneously visualize cRNA after both first and second rounds of amplification, the signal intensity in each lane has been scaled to itself so that lane-to-lane quantitative comparisons are not valid. Background bands in lanes 5 and 6 are system noise that is detected due to the low specific signal obtained from the first round amplification cRNA product.
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