Gene Expression analysis experiments involve the following major steps:
- Experimental Design
- RNA Isolation
- Hybridization to the Chip
- Array Data Analysis
1. Experimental Design:
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.
It is recommended that all experimental treatments be carried out in triplicates to compensate for biological and experimental variation. In vitro experiments using cultured cells should be conducted three different times (not three replicates performed on the same day) strictly following the same experimental procedures. Tumor specimens should be devoid of adjacent tissues and if possible, microdissected to obtain as pure a tumor sample as possible. Cell populations may also be further purified using cell-sorting techniques such as FACS. Dead cells also should be removed by density centrifugation. For comparative gene expression analysis, it is essential that all the experimental conditions such as temperature, CO2, media, reagents, and sample processing be kept identical for all samples.
2. RNA Isolation :
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.Total RNA isolated using TRIzol should be further purified using the Qiagen RNeasy cleanup procedure. The minimum amount of total RNA required for analysis is 500 ng (When RNA amount is limiting, as low as 50 ng of total RNA can be used.) The A260/A280 ratio should be at least 1.8 for pure RNA. The quality of RNA should also be assessed by agarose gel electrophoresis. The Bioanalyzer gel profile should exhibit a 28S band that is 2 times more intense than 18S ribosomal RNA. It is important that the total RNA is free of genomic DNA contamination. We have written Standard Operating Procedures for the isolation of total RNA using both TRIzol and Qiagen RNeasy methods. If genomic DNA contamination is present, it is essential to remove it by DNase treatment, a modification included in the RNeasy cleanup protocol.
3. Target :
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.
4. Hybridization to the Chip :
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.
5. Array Data Analysis:
The images are analyzed using the Beadstudio software. Quality control and data analysis are carried out according to the instructions provided by Illumina. The quality control parameters are as follows:
The intactness of the biological specimen can be monitored by the housekeeping gene controls. These controls consist of probes to housekeeping genes, two probes per gene, which should be expressed in any cellular sample. The housekeeping values for all the arrays in one experiment should be all very similar, otherwise comparison data might not be accurate.
Three types of controls comprise this category: Cy3-labeled Hyb Control, Low Stringency Hyb Control, and High Stringency Hyb Control. The Cy3-Labeled Hyb control consists of six probes with corresponding Cy3-labeled oligonucleotides present in the controls added to the cRNA before hybridization. Following successful hybridization, they produce a signal independent of both the cellular RNA quality and success of the sample prep reactions. Target oligonucleotides for the Cy3 Hyb controls are present at three concentrations (low, medium, and high), yielding gradient hybridization. As with the housekeeping controls, the values for the low, medium and high concentrations for all arrays in one experiment should be very similar. The Low Stringency Hyb Control contains four probes, corresponding to the medium- and high-concentration Cy3 Hyb control targets. In this case, each probe has two mismatch bases distributed in its sequence. If stringency is adequate, these controls yield very low signal. If stringency is too low, they yield signal approaching that of their perfect match counterparts in the Cy3 Hyb control category.
The High Stringency Hyb Control consists of one probe corresponding to a Cy3-labeled oligonucleotide target. The probe/target sequences have a very high G+C content, and should thus hybridize even if hybridization stringency is too high. Hybridizations with too-high stringency are detected on the basis of a signal present from this control in the absence of signal from the other hybridization control probes.
This category consists of probes of random sequence selected to have no corresponding targets in the genomes. The mean signal of these probes defines the system background. This is a comprehensive measurement of background, representing the imaging system background as well as any signal resulting from non-specific binding of dye or cross-hybridization. The BeadStudio application uses the signals and signal standard deviation of these probes to establish gene expression detection limits.