Extraction of RNA from rhino-probe samples : the good, the bad and the ugly

![CDATA[Rhino‐probe™ nasal curettes have been utilized in recent clinical studies to obtain mucosal epithelial cells for in‐vitro studies on the pro-inflammatory effects of diesel exhaust and tobacco smoke. The curettes have been reported to give more consistent sample collection, as well as higher cell numbers of cells than traditional approaches (cytology brushes/lavage). The aim of this study was to develop a Ribose Nucleic Acid (RNA) extraction method for Rhino‐probe™ nasal mucosal cell samples stored in RNAlater. This method will be applied in a study of inflammatory markers in Colorado dairy workers. Nasal epithelial cells were taken from the inferior turbinate with Rhino‐probes™. The samples were transferred to RNAlater and stored at ‐74°C. To process; 500μL of PBS was added, and the samples pelletized by centrifuging. The supernatant was removed and either RLT buffer (Qiagen) or Trizol (Life Sciences) added. The tissues were homogenized for 30s with a motorized pellet pestle. Four RNA extraction methods were tested. Extraction with the RNeasy kit, RNeasy Micro kit and TRIzol, as well as a two‐step homogenization (pestle and QIAshredder) combined with Micro RNeasy kit. A subset of samples were collected and frozen in RLT buffer. These samples were extracted with the RNeasy Micro kit, with and without the QIAshredder step. RNA quantified with a NanoDrop Spectrophotometer. The combination of tissue storage in RLT buffer with two‐step homogenization method provided RNA with the highest concentration and purity, followed RNAlater samples extracted by the same method. RNA yields from the TRIzol method were highly variable and of poor quality. The RNeasy kit yielded very little RNA. The RNAlater is attributed to poor RNA yield due to its viscosity. This viscosity inhibits pellet formation, even with the addition of PBS, leading to sample loss and transfer of RNAlater residue to extraction columns. It is recommended that Rhino‐Probe samples are stored in RNA lysis buffer and snap frozen in the field with dry ice or a freezer cube. For optimal RNA, samples should be processed with commercial extraction kits designed for small tissue quantities and incorporate a two‐step homogenization process such as the pestle and QIAshredder combination.]]