Estimating RNA-quality using GeneChip microarrays.

BMC Genomics
Mario Fasold, Hans Binder

Abstract

Microarrays are a powerful tool for transcriptome analysis. Best results are obtained using high-quality RNA samples for preparation and hybridization. Issues with RNA integrity can lead to low data quality and failure of the microarray experiment. Microarray intensity data contains information to estimate the RNA quality of the sample. We here study the interplay of the characteristics of RNA surface hybridization with the effects of partly truncated transcripts on probe intensity. The 3'/5' intensity gradient, the basis of microarray RNA quality measures, is shown to depend on the degree of competitive binding of specific and of non-specific targets to a particular probe, on the degree of saturation of the probes with bound transcripts and on the distance of the probe from the 3'-end of the transcript. Increasing degrees of non-specific hybridization or of saturation reduce the 3'/5' intensity gradient and if not taken into account, this leads to biased results in common quality measures for GeneChip arrays such as affyslope or the control probe intensity ratio. We also found that short probe sets near the 3'-end of the transcripts are prone to non-specific hybridization presumable because of inaccurate positional assignment ...Continue Reading

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Citations

Jan 12, 2013·Nucleic Acids Research·Andrew HarrisonAlexander E Pozhitkov
Mar 22, 2013·Health Education Research·Berta Murillo PardoLuis García González
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Dec 30, 2017·Oncotarget·Pawel KarpinskiMaria Malgorzata Sasiadek
Oct 28, 2019·Scientific Reports·Malou L H SnijdersMarian C Clahsen-van Groningen

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Datasets Mentioned

BETA
GSM176301
GSM175967

Methods Mentioned

BETA
in vitro transcription
electrophoresis
chip
chips
in-vitro transcription

Software Mentioned

R package affy
R
RatQC
affy package
Affymetrix
affyslope
Bioconductor
AffyRNADegradation

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