Data Validation by Quantitative Real Time PCR (qPCR)

Relative Quantitation of Gene Expression by Real Time qPCR		Validation of Microarray

Microarray analysis performs hundreds or thousands of differentially expressed genes simultaneously while the state of the art technology qPCR provides precise quantification of a wide dynamic range of expression levels due to its increased sensitivity and reproducibility. Our clients can rapidly validate and correlate microarray results for their most exciting research leads. In order to identify real gene specific variation, it is important to normalize gene expression level by carefully selected stable internal control genes. Conventional normalization strategy based on a single constitutively expressed gene that is housekeeping gene leads to erroneous normalization. Therefore, in order to measure expression levels accurately, normalization by multiple housekeeping genes instead of one is required.

Advanced Genomics uses three most stable housekeeping genes as internal controls for calculation of qPCR normalization factor. For better possible outlying values and gene abundance differences between different genes, we use geometric mean instead of arithmetic mean. We perform qPCR to:

• validate gene expression profile of microarray analysis
• validate expression level of full length clones in drug targets
• determine regulatory expression of genes across large population

Note: It is highly recommended that while performing microarray analysis, save 2-5 μg each RNA sample for qPCR validation. It is important to use the same RNA sample for validation by qPCR as is used for microarray analysis because RNA expression profile of biological sample changes dramatically with the change of experiment settings. Tissues stored without stabilization reagent show significant changes in RNA expression. It is important to use RNAlater for stabilizing RNA expression level in tissues.