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Editor—Standard genetic tests usually involve obtaining a blood sample for DNA extraction. Other commonly used sources of DNA are buccal cells1 (from a cheek scraping) and hair roots,2 which are viable alternatives to a blood sample and less invasive to obtain, particularly useful in children. Some genetic tests require full mutation screening (at least of the affected subject in a family) and this can be greatly simplified, particularly for large, multiexon genes, by screening the mRNA as opposed to the DNA.3 The reason for this is simple; the mRNA contains all the relevant sequence in a compact “ready to screen” package rather than the many discrete packages (exons) found in DNA. In addition, mRNA is one step further down the gene-protein pathway and can yield more relevant information on the effect of a mutation (at least with regard to splicing). The problem with using RNA as the primary source for mutation analysis has been that it is rather less stable than DNA and is easily degraded both in vivo and in vitro by endogenous and exogenous ribonucleases. In order to extract it from blood, it is necessary to separate the lymphocytes from the red blood cells before following standard extraction procedures. This, coupled with the fact that the blood must be fresh (ideally no more than 24 hours old), makes the use of RNA extracted from blood a labour intensive and awkward route to mutation detection.
A compounding difficulty in RNA screening is that the gene of interest may only be expressed at very low levels in the tissue analysed, for instance, lymphocytes. This can be circumvented, at least in some cases, by the use of two rounds of PCR (“nested PCR”) to amplify trace quantities of the appropriate message (known as “ectopic transcripts”).4 However, we have found this to be unreliable for the COL4A5 gene, in which mutations are responsible for Alport syndrome.5Consequently, we sought an alternative but easily accessible source of RNA. Hair roots, although widely used for DNA, seem to have been overlooked as a source of RNA. We used off the shelf proprietary RNA extraction kits (“RNA Isolator” from Genosys) to obtain RNA from between 10 and 20 hair roots. The RNA was resuspended in 30 μl of RNAse free water and 1 μl used for RT-PCR (using the Access-RT-PCR kit from Promega). As can be seen in fig 1, the amplification of four overlapping segments that cover the COL4A5gene is clear evidence that the RNA is of good quality (and quantity) to make mutation screening for this large and complex gene much easier. Mutations have already been detected in these RT-PCR products6 after screening with fluorescent chemical cleavage of mismatch.7 We have also had success amplifying the ectopic transcripts of the factor VIII gene. Thus, at least for these two genes, hair roots serve as a good material for RNA analysis. We do not know whether the reason for improved RT-PCR is that hair roots have wider gene expression than lymphocytes or that they have higher levels of ectopic transcription.
Hair roots are easy (and cheap) to transport nationally or internationally and are not considered to be at high risk of viruses in the same way that blood is. We have successfully extracted RNA from hair roots that had been at room temperature for up to 10 days after plucking, suggesting that the RNA is better protected in the hair root cells than in blood (which does not yield RNA after this length of time). Our experience suggests that it is immaterial whether the hair is grey or artificially dyed, although yield is lower from fine hair than from thick hair. Eyebrow hair and chest hair have also yielded good RNA (so baldness is no obstacle!). Plucking individual hairs is virtually painless and so is less distressing for young children. We believe that the ease of obtaining hair roots, the increased stability of the RNA, and the ease of RNA preparation may make this the ideal source for many mutation detection projects.
This work was supported by the National Kidney Research Fund.
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