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Title: Sequence polymorphism can produce serious artifacts in real-time PCR assays: lessons from Pacific oysters

Author
item LANG, R - OREGON STATE UNIVERSITY
item Camara, Mark

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2008
Publication Date: 5/20/2008
Citation: Taris, N.G., Lang, R.P., Camara, M.D. 2008. Sequence polymorphism can produce serious artifacts in real-time PCR assays: lessons from Pacific oysters. BioMed Central (BMC) Genomics.9:234.

Interpretive Summary: Quantitative real time PCR has become a routine approach to quantify levels of gene expression, and is often used to validate whole-transcriptome approaches such as suppressive subtractive hybridization (SSH) or differential display. We examined the expression levels of two different genes that preliminary results using cDNA-AFLP indicated are differentially expressed in response to bacterial infection in the Pacific oyster, Crassostrea gigas, using a several primer sets, all of which were technically acceptable using standard optimization criteria. Surprisingly, different primer sets produced completely different results with different biological implications. Sequence analysis revealed that polymorphisms in the sequences studied are responsible for these inconsistent results because they impact the ability of specific primers to amplify the target sequence. This problem is similar to null alleles in standard PCR. We conclude that despite efforts to optimize the methodology, misleading results are still possible, even when standard optimization approaches are followed, and that more rigorous testing is necessary in order to be confident of results obtained using quantitative real time PCR.

Technical Abstract: Since it was first described in the mid-1990s, quantitative real time PCR (Q-PCR) has been widely used in many fields of biomedical research and molecular diagnostics. This method is routinely used to validate whole transcriptome analyses such as DNA microarrays, suppressive subtractive hybridization (SSH) or differential display techniques such as cDNA-AFLP (Amplification Fragment Length Polymorphism). Despite efforts to optimize the methodology, misleading results are still possible, even when standard optimization approaches are followed.