GeneChip Expression Analysis Experiments involve the following major steps:
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, should be 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.
RNA isolation. The quality of the RNA is the single most important determinant of success of a 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 also 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.
The A260/A280 ratio should be at least 1.9 for pure RNA. The quality of RNA should also be assessed by agarose gel electrophoresis. The agarose gel profile should exhibit a 28S band that is 2 times more intense than 18S ribosomal RNA (Figure 4). It is important that the total RNA is free from genomic DNA contamination. There are precautions to be taken while using the RNeasy kit to avoid genomic DNA contamination. We have written Standard Operating Procedures for the isolation of total RNA using both TRIzol and RNeasy methods and these are available to all facility users upon request. If genomic DNA contamination is present, it is essential to remove it by DNase treatment. DNase then can be removed by heat inactivation followed by RNeasy cleanup using Qiagen kit. We also strongly encourage everyone to use the Agilent Bionalyzer.
The minimum amount of total RNA required for GeneChip analysis is 5 ug.
Target cRNA Preparation. Good 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 poly (A) RNA present in the starting total RNA sample. Single stranded cDNA is then converted into double stranded cDNA which is extracted with phenol/chloroform and then precipitated with ethanol. An in vitro transcription (IVT) reaction is then carried out in the presence of biotinylated UTP and CTP to produce biotin-labeled cRNA from the double stranded cDNA. cRNA is then fragmented in the presence of heat and Mg++ before hybridization to the test arrays.
Due to the high cost of cDNA and cRNA synthesis reactions, we use a very strict quality control measures during the target preparation procedures (See Figure.4). At each step the quality of sample is accessed by Agilent Bioanalyzer.
Test array hybridization. Test arrays are relatively inexpensive and are used as an assessment tool for determining target quality and labeling efficiency. Fragmented cRNA is hybridized to the test array for 16 hours at 450 C. The test array is then washed and stained with streptavidin-phycoerythrin using the fluidics station and then scanned using GeneArray scanner.
- Test array data analysis. Images will be analyzed by following quality control parameters as provided by Affymetrix:
- 3'/5' ratio of housekeeping genes. This is a measure of the efficiency of the cDNA synthesis reaction. Reverse transcriptase synthesizes cDNA starting from the 3'-end of an mRNA and ending at the 5'-end. All Affymetrix arrays contain probes for the regions corresponding to 3', middle and 5'-end of the house keeping genes such as GAPDH and Actin. The ratio of signal intensity for 3' probes to that from 5' probes provides a measure of the number of cDNA synthesis reactions that went to completion (full-length cDNA is synthesized). An ideal ratio would be 1 whereas a higher value indicates that many cDNAs were started but did not go to completion. The 3'/5' ratio for the housekeeping genes should be at most 3' If the ratio is above 3, some sensitivity of the assay may be lost.
- Presence of spiked control cRNAs. Bio-B, C, D and CRE serve as a controls for hybridization and are spiked at the following concentrations: BioB: 1.5 pM, BioC: 5.0 pM, BioD: 25.0 pM, BioCRE: 100 pM. We specifically look at the average difference values which should be present in increasing amounts, B being the least and CRE the highest
- Background values with standard deviation. Background value is a measure of the signal intensity caused by autofluorescence of the array surface as well as nonspecific binding of target or stain molecules (SAPE). The background values for all the arrays in one experiment should be very similar to each other; otherwise comparison data may not be accurate. Non-specific binding causes a low signal to noise ratio, which means that genes for transcripts present at very low levels in the sample may incorrectly be called absent. Thus, high background creates an overall loss of sensitivity in the experiment.
- Q value. Noise (Q value) results from small variations in the digitized signal observed by the scanner as it samples the probe array.s surface. It is measured by examining pixel-to-pixel variations in background intensities. The noise value for all the arrays in one experiment should be very similar to each other.
- Scaling factor. The scaling factor provides a measure of the brightness of the array. The .brightness. (image intensity) varies from array to array. Non-biological factors (amount and quality of the cRNA, amount of stain, or other experimental variation) can contribute to the overall variability in hybridization intensities. In order to reliably compare data from multiple arrays, it is essential that the intensity of the arrays be brought to the same level. Scaling is a mathematical technique used by the Microarray Suite Software (MAS) to minimize differences in overall signal intensities between two or more arrays, thus allowing for more reliable detection of biologically relevant changes in the same sample. MAS calculates the overall intensity of an array by averaging the intensity values of every probe set on the array with the exception of the top and bottom 2% of the probe set intensities. The average intensity of the array is then multiplied by the scaling factor to bring it to an arbitrary target intensity value (usually 500) set by the user. Thus, scaling allows a number of experiments to become normalized to one target intensity, allowing comparison between any two experiments. In a particular set of experiments, the Scaling Factor value for all the arrays should be very close to each other (within three-fold of each other).
Hybridization to the Standard array
If the data obtained from the test array is satisfactory, the sample is hybridized to the standard array for 16 hours at 45 0C. The standard array is then washed and stained using the fluidics station and then scanned. The images will be analyzed using Microarray Suite Software and comparison analysis will be carried out according to the instructions provided by Affymetrix.