Gene expression DNA chips are a set of DNA sequences (probes) arrayed on to a solid substrate at ultra-high density (10.000 - 1.3 million sequences per chip), thus they are also known as DNA microarrays. The DNA chips constitute a very powerful tool within the genomic era, because they enable investigators to simultaneously monitor gene expression of a very large number of genes relevant to a particular biological phenomenon.

There are different kinds of DNA chips and at our microarray facility we are able to generate cDNA microarrays. This kind of DNA chips is very important for species which lack elaborated genomic tools ("orphan" species regarding genomic tools), because they can be generated without any previous genome sequence information. In consequence, at the DNA microarray facility of the Biotechnology and Agrobiodiversity laboratory at CIAT, we have been able to generate cDNA microarrays for gene expression analysis in Brachiaria spp. and Manihot esculenta for different phenomena.

How to build a dedicated cDNA microarray for an "orphan" species?

An ideal dedicated cDNA microarray is a microarray that contains the full set of genes relevant to a certain biological phenomenon. To approach this ideal, we build cDNA microarrays from subtractive cDNA libraries enriched in the sequences present at the specific tissue under the target condition. This way we have developed the following cDNA microarrays at the Biotechnology and Agrobiodiversity laboratory at CIAT:

Using the former cDNA microarrays or from other molecular biology labs in a few projects, we identify differentially expressed sequences between control plants (or bacteria) and the corresponding treatments.

Subsequently, we sequence the cDNA library clones that have the sequences with interesting expression patterns, and we find their putative functions using the public sequence databases (such as EMBL, GenBank, SWISS-PROT, and alike) supported by various Bioinformatics tools.

Following, you will find the methods we follow for every step of the gene expression analysis carried out at the Biotechnology and Agrobiodiversity laboratory at CIAT, and you can also access the respective protocols. Our protocols come from the adaptation of protocols found publicly, either as scientific papers, or at the web sites of well-know molecular biology laboratories such a TIGR.

cDNA libraries

To obtain the full set of genes involved in a certain biological phenomenon we isolate RNA using the most convenient column-based commercial kit (Promega, QIAGEN, Arcturus). Then we synthesize cDNA using the SMART™ PCR cDNA Synthesis Kit from Clontech, and we generate the relevant subtractive cDNA libraries with Clontech PCR-Select™ cDNA Subtraction Kit and the pGEM®-T Easy Vector System from Promega. For normalization purposes we also clone non-subtracted cDNA from the unsubtracted controls of the subtraction kit.

Finally we transformed E. coli and pick a relevant number of colonies into 384-well culture plates.

Microarray construction

• Probe preparation. Adapted from "Manufacturing DNA microarrays from unpurified PCR products"
• Microarray spotting and preparation

Sample labeling and hybridization

Adapted from "A robust method for the amplification of RNA in the sense orientation".

First, total RNA is amplified by in vitro tanscription, to keep original gene expression patterns, either in sense or anti-sense orientation. Then the amplified RNA is labeled by the indirect method.

Finally the labeled samples are hybridized to the Microarray.

Microarray scanning

We use the VersArray ChipReader from Bio-Rad to scan the microarrays