DNA microarrays

sample quality control, array hybridization and scanning.

Elva Diaz, Gustavo A. Barisone

Research output: Contribution to journalArticle

Abstract

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number49
StatePublished - 2011

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Microarrays
Oligonucleotide Array Sequence Analysis
RNA
Quality Control
Quality control
DNA
Scanning
Transcription
Color
Genes
Tissue
Lab-on-a-chip
Complementary RNA
Neurology
Nucleotides
Brain Neoplasms
Labeling
Nervous System
Reverse Transcription
Amplification

ASJC Scopus subject areas

  • Medicine(all)

Cite this

DNA microarrays : sample quality control, array hybridization and scanning. / Diaz, Elva; Barisone, Gustavo A.

In: Journal of visualized experiments : JoVE, No. 49, 2011.

Research output: Contribution to journalArticle

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