Editor,—Sarela and colleagues ( Gut 2000 >46: 645–50 OpenUrlCrossRefPubMedWeb of Science) report on the association of Survivin gene expression and prognosis in recurrent colorectal cancer. The methods described for detectingSurvivin mRNA relied on reverse transcription-polymerase chain reaction (RT-PCR), an exquisitely sensitive technique that has not previously been validated for this gene. We wish to point out three areas of technical difficulty in the methodology.

(A) The fidelity of mRNA extraction and RT was tested using oligonucleotide primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a “housekeeping” gene. However, this may give rise to false positives by amplification of pseudogenes from contaminating genomic DNA.1 The β-actin primers (as described by Raff and colleagues1) do not amplify genomic DNA and therefore provide absolute evidence that RT has been successful. Alternatively, this problem could be corrected either by DNase digestion of RNA before RT or by having negative RT controls for each sample.

(B) The process of RT using an oligo dT nucleotide as the RT primer results in the creation of …

Professor P J Guillou, Professional Surgical Unit, Level 8, Clinical Sciences Building, St James's University Hospital, Leeds LS9 7TF, UK.p.j.guillou{at}leeds.ac.uk