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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

  • The Tissue Typing Laboratory tests for compatibility between patients who require an organ or bone marrow transplant and potential donors. In the case of bone marrow transplantation it is important that the tissue type of patient and donor are matched to minimize graft versus host disease. In organ transplantation matching patient and donor tissue types reduces the risk of chronic rejection caused by the development of donor-specific antibodies in the patient against tissue-type mismatches.

    A person’s tissue type comprises a set of highly polymorphic proteins called human leukocyte antigens (HLA), which are found on the surface of most cells. HLA plays a central role in adaptive immunity, generating an immune response to pathogens but simultaneously presenting a barrier to marrow and organ transplantation. PCR-based DNA methods have largely replaced serology in tissue typing and, like most laboratories testing large numbers of clinical samples, automation is becoming the norm. In 2006 the New Zealand Tissue Typing Laboratory started using Roche’s MagNA Pure Compact robot to automate DNA extraction.

    DNA sequencing has revealed potentially important polymorphisms spread over several exons which, in genomic DNA, can be separated by several kilobases. Tissue typing is becoming an increasingly time-consuming and expensive process using genomic DNA.

    We decided to investigate the use of the MagNA Pure robot to isolate total RNA from blood, synthesize cDNA using a Transcriptor cDNA Synthesis kit, and for use as a template for HLA sequencing. RNA is a simpler template than genomic DNA because there are no introns. A random selection of blood samples from transplant patients and donors was selected; these had already been HLA typed using genomic DNA to allow a comparison.

  • Materials and Methods

    Samples and extractions

    RNA was extracted from 64 random uncoagulated citrate phosphate dextrose (CPD) blood samples using the MagNA Pure Compact RNA Isolation Kit. The samples were from transplant patients and healthy donors and the blood had been collected from 8 hours prior to extraction, and up to 14 days prior to extraction. Blood samples not extracted immediately were stored at 4°C. Prior to extraction blood samples were thoroughly mixed by several inversions. RNA was quantified using a Nanodrop spectrophotometer.

    cDNA Synthesis

    In all cases cDNA was synthesized from 0.1 µg (10 µL) RNA using the Transcriptor cDNA Synthesis Kit with random hexamers and oligodT primer.

    HLA Amplification and HLA typing

    HLA genes, HLA-A, -B, -DQB1, and DRB1, were PCR amplified with AmpliTaq Gold using locus-specific exon primers in exons 1 and 5 (HLA-A and -B) or exons 1 and 4 (HLA-DQB1 and -DRB1).

    Nucleotide sequences of amplified exons were determined by cycle sequencing (BigDye Terminator chemistry). Sequenced products were separated by capillary electrophoresis using a 3130XL Genetic Analyzer. Nucleotide sequences were compiled and HLA types assigned using SBTengine, dedicated software that compares heterozygous nucleotide sequences with a library of HLA alleles.

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