Gene Expression analysis experiments involve the following major steps:
It is absolutely essential to plan and execute the experiments with utmost care. It is important to begin the planning of the microarray experiment with a proper question. The experimental model/system should be well-characterized or well-defined with an independent experimental verification. For example, if a growth factor was added which induces differentiation in 24-48 hours, but you are collecting RNA at three hours post-treatment, you should still check a parallel culture for verification that differentiation occurred at the 24-48 hours period. If possible, a quick check for a gene that is known to be affected by the treatment should be performed.
The quality of the RNA is the single most important determinant of a successful GeneChip analysis assay. Particularly, differential degradation of RNA can lead to erroneous conclusions about both the relative and absolute mRNA levels in the specimens. Although either mRNA or total RNA can be used as starting material, we prefer total RNA for two reasons: (1) isolating total RNA is easier and more economical than isolating mRNA and (2) there is loss of starting material during mRNA purification and consequently more mRNA is required to achieve sensitivity similar to that of the total RNA. In addition, there may be differential loss of individual mRNAs. We recommend TRIzol reagent for isolation of total RNA from tissue specimens as well as cultured and blood cells. Total RNA isolated using TRIzol should be further purified using the Qiagen RNeasy cleanup procedure.
High quality total RNA is used as starting material to obtain labeled cRNA. In the first step, single stranded cDNA is synthesized by reverse transcription using the poly (A) RNA present in the starting total RNA sample. Single stranded cDNA is then converted into double stranded cDNA and purified using the Illumina TotalPrep RNA Amplification Kit. An in vitro transcription (IVT) reaction is then carried out overnight in the presence of biotinylated UTP and CTP to produce biotin-labeled cRNA from the double stranded cDNA. The cRNA from the IVT reaction is purified using the same Amplification Kit. Due to the high cost of cDNA and cRNA synthesis reactions, we use very strict quality control measures during the target preparation procedures.
After the quality control assessment, 1.5 µg of cRNA is mixed with the hybridization controls and it is hybridized to the array. The array is hybridized for 16 hours in a hybridization oven with a rocking platform at 58°C. The array chip then goes through a series of washes before it is stained with streptavidin-Cy3. After the staining, it goes through a final wash and drying. The array is scanned using the Illumina BeadArray reader.