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Editor,—Sarela and colleagues (
) 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 cDNA templates for all mRNAs in the sample. This may be a problem if the gene for effector cell protease receptor 1 (EPR-1) is expressed. This gene codes for a cellular receptor of blood clotting factor Xa.2 The DNA sequence for this gene is highly homologous to that of Survivin and differs by only five nucleotide changes and six nucleotide insertions.3 The reverse primer described recognises the EPR-1 sequences (as ascertained by searching of the basic local alignment search tool of the National Cell Biology Institute (BLAST)). The forward primer does not produce a match on BLAST searching but only 1011 bases of the sequence for EPR-1 have been published on Genebank (GeneBank Accession No. L26245. Human effector cell protease receptor-1 (EPR-1) mRNA, partial CDs). Implicit in the description is that this sequence is incomplete. Given the close similarity between the probable sequences of the two genes it is not impossible that this homology continues and could provide a recognition site for the forward primer in EPR-1. This problem has been alluded to by Mahotka and colleagues4 who used a sequence specific RT primer to eliminate it but was not taken into account elsewhere in work on survival in small cell lung cancers.5 This may explain the detection of “Survivin” mRNA in normal colorectal mucosa.
(C) The PCR primers as published are in the first and fourth exons. The amplified sequence would be expected to include the published splice variants caused by deletion of the third exon or insertion of the 2B exon, as described by Mahotka and colleagues.4 This would result in multiple bands detected on agarose gel. We would be interested to know whether these points were taken into account.
Editor,—We thank Miller and colleagues for their interest in our study, and for pointing out the areas of technical difficulty with reverse transcription-polymerase chain reaction (RT-PCR) based projects.
(A) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification is well established as a control for the fidelity of RT and has been used as such in numerous studies, including that of Mahotka and colleagues1-1 quoted by Miller et al. In our cell culture experiments, the intron spanning GAPDH primers used in the present investigation yielded more consistent results than β-actin primers. While GAPDH pseudogenes may occasionally be problematic, the modified Catrimox RNA isolation technique used in the present and other studies from our laboratory1-2 results in minimal genomic DNA contamination, as confirmed by RT negative controls.
(B) Miller et al fail to recognise that although the genomic sequence of effector cell protease receptor 1 (EPR-1) is highly homologous to Survivin,northern hybridisation with single strand specific probes has identified distinct and mutually exclusive transcripts forSurvivin (1.9 kb) and EPR-1 (1.3 kb).1-3 Consequently, even if we were to accept Milleret al's unsupported hypothesis regarding a recognition site for the Survivin forward primer in EPR-1, it is highly unlikely that an EPR-1 product of the same size and sequence as Survivin would be amplified. The specificity of our RT-PCR data is further confirmed by immunohistochemical analysis (using a monoclonal antibody kindly provided by the Yale group) that demonstrates a similar prevalence ofSurvivin protein expression, and a strong degree of concordance between protein and mRNA expression, in colorectal cancer.1-4
(C) Survivin splice variants, which were described in renal cell carcinoma cell lines1-1 after our paper was accepted for publication, are certainly intriguing. On agarose gel electrophoresis we noted the expectedSurvivin amplification product of 338 bp (confirmed by direct sequencing) as the prominent band in all cases that were scored Survivin positive. In a small proportion of cases, additional minor bands, which may have resulted from alternative splicing, were noted. As discussed by Mahotka and colleagues,1-1 alternative splicing adds considerably to the complexity of systems controlling apoptosis. Further investigation of the significance of this phenomenon in colorectal cancer is underway.