RNA Isolation

Extraction of total RNA from biological fluids can be challenging, as typically these samples are rich in protein and lipids and low in RNA content. Nevertheless, several protocols were developed that include lysis using a chaotropic salt, such as guani- dinium thiocyanate that simultaneously solubilises the biological material and denatures proteins followed by a solid phase extraction procedure on silica columns. Removal of proteins and lipids results in elution of high-quality RNA (Turchinovich et al. 2011). Recently, commercially available products that can be used to isolate RNAs smaller than 1000 nucleotides have been introduced specifically for RNA isolation from biofluids. Based on spin columns that exploit the use of resins and thus do not require phenol or chloroform extraction, these protocols can be applied to obtain high-quality RNA. However, despite the improved purity, all the extraction methods have low RNA yield. Thus, assessing the concentration using OD260 absorbance is inaccurate. Therefore, a fixed volume of eluted RNA is typically used for all downstream applications (Zampetaki et al. 2010). The efficiency of RNA extraction can improve with the inclusion of a carrier, such as glycogen or bacteriophage MS2 (Andreasen et al. 2010; Turchinovich et al. 2011). Perhaps more importantly, in large clinical studies, the use of a carrier may improve the consistency of RNA extraction across samples.

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