RNA quality of testicular tissues after different fixation protocols, storage and staining methods.

<p>The RNA quality was determined on an Agilent Bioanalyzer where higher RIN values reflect better RNA quality. Error bars show standard deviations. The statistical significance was evaluated by Student's t-test, * p<0.05; ** p<0.01. A: Frozen vs paraffin embedded tissues. Quality of RNA, purified from frozen sections (blue n = 9) or from tissues fixed in either formalin (green, n = 15), Stieves (red, n = 15) or GR fixative (yellow, n = 15) and embedded in paraffin. B: Storage of frozen sections. RNA quality in seminoma and rat testis after storage for 1, 3, 7 or 30 days as frozen sections at −80°C (blue, n = 8); storage at −80°C in 100% ethanol (green, n = 8); or sections freshly cut from a tissue sample that was repeatedly removed from −80°C (red, n = 8). C: Time-dependent RNA degradation after staining. Sections were fixed in 75% ethanol or 60% isopropanol, and stained according to the respective protocols (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005536#pone-0005536-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005536#pone-0005536-t003" target="_blank">3</a>). The RNA quality was assessed just before staining (BS) and 0, 4, 24 and 72 hours after staining. * and ** show that for the NBT BCIP and Kiel protocols, the RNA quality was significantly reduced already during staining (between BS and time 0) and * indicate significant differences during the subsequent storage (between 0 and 72 hours).