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PA-320 Optimisation of RNA extraction methods for small blood volumes for future use in TB host transcriptomic biosignature applications
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  1. Monique von Molendorff,
  2. Novel Chegou,
  3. Tracey Jooste
  1. DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University, South Africa

Abstract

Background Whole-blood-based transcriptomic methods have limitations, including in the amount of blood required for standard RNA blood collection tubes. This is particularly relevant to individuals with difficulty in providing large volumes of blood, such as children. Furthermore, the cold chain required for storing blood tubes is problematic in field applications and in remote settings. It is therefore important to optimise RNA extraction techniques to allow for the isolation of high quantity and quality RNA from small blood volumes, and samples that are easier to collect and store under field conditions. Thus, the aim was to evaluate the quantity and quality of RNA extracted from small volumes of whole blood including dried blood spots (DBS) using three commercial extraction kits, to determine suitability for future use in RT-PCR-based experiments.

Methods Total RNA was extracted from small blood volumes (500μl, 100μl, 50μl) and DBS samples using the GenElute™ Total RNA Purification, PureLink® RNA Mini, and Tempus™ Spin RNA Isolation Kits. The yield, purity and integrity of the resulting RNA was assessed with fluorometry, spectrophotometry and agarose gel electrophoresis respectively.

Results An average of 2321 ± 456.30 ng and 336.3 ± 113.6 ng RNA was obtained from 500 μl of blood with the Tempus™and PureLink® kits respectively. Overall, the RNA isolated with both kits were intact and of high quality. RNA isolated from lower blood volumes (100 μl, 50 μl and DBS) using all three kits, was not of sufficient quantity or quality.

Conclusion Preliminary findings indicate that the quantity and quality of RNA isolated from 500μl of blood using either the PureLink® or Tempus™ kits may be sufficient for downstream transcriptomic analysis. Upon completion, our findings may be valuable in future studies that are conducted in individuals with difficulty in providing large volumes of blood such as children.

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