Right to Know
Transcriptional expression profiling in the MASR is performed in one of two modes to derive the maximum amount of information in the most cost-effective manner. “Genome-wide” expression profiling is first used when the fingerprint pattern and the identity of all possible expressed genes are sought. “Targeted” expression profiling is performed following genome-wide screening analysis, and permits focus on a large number of replicated conditions, time points, cancer stages, or samples from different individuals.
Analysis of tens of thousands of genes is a new challenge in molecular biology and requires a very careful design of experiments when researchers need high quality results. Basically any kind of technical variation will be observed in the results. When samples shall be compared for differential gene expression, every technical step must be done as similar as possible.
Here are some examples of how results can be compromised:
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Cell cultures are treated slightly different (cell density, change of medium, different batches of FCS). Induction of stress response can be observed, changes of cell cycle or apoptosis.
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Samples are treated different before RNA isolation (storage, freezing). Heat shock or cold shock can be induced, apoptosis or RNA degradation can appear.
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Alterations in RNA isolation protocols (thawing of samples, usage of different methods of RNA isolation within a project, usage of mRNA and total RNA within a project. Differential gene expression can be observed in hundreds of genes.
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Differences in RNA integrity due to technical variations (we can quantify RNA integrity of your samples, see RNA analysis) Stable mRNAs behave like upregulated, unstable RNAs downregulated
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Variations in chip processing (different batches of enzymes, several people doing the experiments). When minor alterations in gene expression shall be interogated, these differences can completely camouflage biological differences.
Sample Quality and Quantity Requirement
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mRNA expression profiling: 5~10 ug of intact total RNA in DEPC-H2O at the concentration 0.5ug/ul needed.
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microRNA expression profiling: 5~10 ug non-column purified total RNA (0.5~1.0 ug/ul) in DEPC-H2O needed.
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SNP genotyping on Affymetrix Chips: 0.5~1.0 ug of genomic DNA needed
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CGH: 5~10 ug genomic DNA needed.
