High-density RPPAs require a large number of high-quality human biospecimens, which must be banked at reputable institutes, contain a high percentage of tumor, include comprehensive clinical data, and be verified by a certified pathologist. Such samples are often expensive and hard to find.
OriGene’s cancer biorepository of 12,000 donors allows its researchers to use a large number of samples across 11 cancers for comprehensive cancer RPPAs.
Another challenge is extracting and standardizing a large number of samples from various tissues and different tumor content. The most straightforward solution is using a single standard operating procedure for all samples, regardless of tumor content.
Some researchers use laser-capture microdissection to enrich the sample for only cancer cells. Although this method is more accurate, it is often unnecessary and results in limited and expensive material. Mild nondenaturizing detergents (NP40 and Deoxy-cholate) with complete protease and phosphatase inhibitor cocktails are preferred in the extraction process in order to preserve the structure and phosphorylation state of the proteins.
Quantifying the different lysates remains a problem. The most commonly used method is protein concentration, in which all samples are diluted to a single concentration (usually 1 mg/mL), followed by 4–5 serial dilutions, which provides a single standardized curve. This curve is printed in triplicate for more accurate quantification.
This procedure is sufficient to accurately detect quantitative changes among samples (Figures 1B and 2). OriGene has found an 84% match between IHC data in the patient pathology report and the expression level measured by the array for ERBB2 among breast samples. These were further confirmed by Western blot analysis.