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Alghali, AlAnazi, and AlGhadam: RNA purification from saliva for biodosimetry applications

Introduction: The work presented here is to assess the possibility of using saliva instead of blood samples to produce a less invasive method of collecting RNA. This can be used to measure gene expression and then used in a biodosimetry device to rapidly measure expression levels of a low-density gene set that can define radiation exposure, dose and injury in a public health emergency.

Objectives: Assess the possibility of using saliva instead of blood to produce a less invasive method of collecting purified RNA for biodosimetry studies.

Materials and methods: Saliva (200 µl) was collected from a donor and immediately mixed with RNAprotect saliva reagent to stabilize the salivary RNA. Three types of sample were prepared: (i) saliva mixed with RNAprotect just before RNA extraction, (ii) saliva mixed with RNAprotect and incubated for 24 hours before RNA extraction and (iii) saliva in culture for 24 hours (37°C; 5% CO2) then stabilized with RNAprotect. Total RNA was purified from stabilized saliva using the RNeasy Micro Kit (Qiagen, Hilden, Germany). RNA concentration was determined by using Epoch Instrument Spectrophotometer (BioTek Instruments, Winooski, VT, USA). RNA quality was determined by RNA 6000 Pico Assay Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Gene expression analysis was performed by chemical ligation-dependent probe amplification (CLPA).

Results: Using gel electrophoresis on our saliva samples we found the two ribosomal subunits (28S, 18S) which are required to perform CLPA (Figure 1).

FIGURE 1

Electrophoresis gel from saliva samples. Both ribosomal subunits 18S and 28S are observed in the non-cultured samples 2 and 4 and in the cultured samples 9 and 10.

Suppl-1-36-fig1.jpg

Conclusions: We have shown that it is possible to purify RNA from saliva as demonstrated in our samples. Although we did not get enough RNA to be able to detect a signal with the CLPA we believe that once the protocol for RNA purification is optimized we will be able to run these samples with CLPA for better gene expression analysis.

Acknowledgements: This study was conducted at The University of Arizona College of Medicine Centre for Applied NanoBioscience and Medicine, Phoenix, AZ, USA, sponsored by Al-Imam Muhammad ibn Saud University summer research programmes.




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