Microarray technology is used to gain insight into the expression levels of mRNAs or small RNAs of the entire transcriptome.
How to start?
The very first step when considering microarray experiments is to clarify objectives and determine experimental design. This can be done with experts at the Genomic Analysis Lab and at the Bioinformatics Unit at TAU. A meeting can be scheduled to ask the proper questions and help construct an effective design using the available microarray facilities.
- Choosing Samples
Microarray analysis of gene expression changes is very sensitve. It is advisable to compare samples that are initially similar. Examples:
- Knock-out animal models
- Tissue comparisons
- Drug treatments
- Stably transfected cell lines
- How many replicates are needed?
As many as possible! Three BIOLOGICAL replicates are usually sufficient for cell-line gene expression, but more samples may be needed for individual cell gene expression.
- If the data is particularly noisy (e.g. samples from very small numbers of cells), more replicates may be needed.
- Do equal numbers of replicates for each condition/comparison to keep the later analysis simple.
- Use independent samples.
- Technical replicates are not necessary.
- Preferably, samples should not be pooled, as every sample is already a pool and contains mRNA from many cells. If cell number obtained from each individual is very small, pooling is the best way of reducing the noise while keeping the number of hydridizations reasonably small. However, it is important to pool the biological material (tissue, cells), and not the purified RNA or labeled cDNA.
- Excellent RNA quality is needed, never include any sample that looks suspicious.
- Recommended amounts and concentration of RNA samples (for 1 chip):
|Type of Chip||Amount of RNA needed|
|All exon expression assay||2-3 µg (concentration above 0.5 µg/µl)|
|miRNA assay||2-3 µg (concentration above 0.5 µg/µl)|
Excellent RNA quality is an essential parameter for the success of the Microarray assay. It is recommended that the RNA isolation methods will be TRIzol Reagent (Invitrogen) followed by clean-up with Phase Lock Gel (PGL tubes) and precipitation. Alternatively, the cleanup could be with QIAGEN RNeasy Mini Kit. If the RNA is intended for miRNA assay, be sure that you purchase a kit designed for small RNA, such as TRIzol.
RNA quality and quantity should be checked by running an RNAse free, 1% agarose gel and QC analysis by NanoDrop or BioAnalyzer (NanoDrop analysis is available at the sequencing unit, Life Science, TAU). The Spectrophotometeric absorbance parameters of the sample should be: 260/280= 1.8-2.0 and 260/230>2.0.
Once RNA extraction is completed, aliquot the samples in several 1.5 ml tubes and store at -80ºC in order to avoid repeated thawing-freezing cycles.
- Replicates: We recommend performing 3 biological replicates for each treatment.
- RNA quality and quantity: check RNA by running on RNAse free, 1.2% agarose gel, and QC analysis by NanoDrop. The Spectrophotometric absorbance parameters of the sample should be: 260/280=1.8-2.0 and 260/230>1.8.
- For more technical details regarding RNA extraction, quantities and quality, please contact Orly at firstname.lastname@example.org
For mRNA and miRNA expression arrays (prices are per sample):
|TAU users||Other academic users|
|Chip price (Eisenberg Bros.)||$220||$220|
|Reagents and lab. work||$330||$405|
* come 4 in one strip ($880 for one strip), VAT included
The preliminary analysis from raw datafiles is performed on various platforms such as the Partek Genomics Suite. Further analysis of differentially expressed gene-lists function and pathways is performed using various updated web-based tools. The analysis is generally done on a collaborative basis. For more details, please contact Dr. Metsada Pasmanik-Chor: email@example.com , 03 640 6992.