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March 01, 2011 (Vol. 31, No. 5)

Sequencing-Based HLA Typing Using RNA

Advances in Extractin and Enzymology Open Door for RNA as Alternative to Genomic DNA

  • Conclusions

    Advances in extraction technology and enzymology mean that RNA now offers a realistic and simpler alternative to genomic DNA in the Tissue Typing Laboratory. The RNA method will also be more cost effective. The method can be simplified further by including cDNA synthesis and PCR in one tube. The method should also be amenable to automation after RNA extraction.

    HLA genes are readily amplified from cDNA, sequenced, and typed from the cDNA (most are heterozygous). The tissue types are in agreement with the DNA-based types for these samples. With no introns this method offers a much simpler sequencing strategy with only 2–4 sequencing reactions compared to 8–12 when genomic DNA is used as template.

    Using RNA as a template for HLA typing is also a useful tool for checking for the expression of HLA alleles, which may be missed using genomic DNA. We have used this approach to characterize a new null allele, HLA-A*01:34N, which has a novel splice site in exon 4. Genomic DNA sequencing showed only a silent substitution in exon 4 (GCG instead of “normal” GCA) but sequencing cDNA showed that exon 4 transcribed from this allele started after this SNP, as it had created an alternative splice site.

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