RNA Extraction Method Impacts Quality Metrics and Sequencing Results in Formalin-Fixed, Paraffin-Embedded Tissue Samples.

Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are being increasingly used in molecular cancer research. Compared with fresh-frozen tissue, the nucleic acid analysis of FFPE tissue is technically more challenging. This study aimed to compare the impact of 3 different RNA extraction methods on yield, quality, and sequencing-based gene expression results in FFPE samples. RNA extraction was performed in 16 FFPE tumor specimens from patients with diffuse large B-cell lymphoma and in reference FFPE material from microsatellite-stable and microsatellite-instable cell lines (3 replicates each) using 2 silica-based procedures (A, miRNeasy FFPE; C, iCatcher FFPE Tissue RNA) and 1 isotachophoresis-based procedure (B, Ionic FFPE to Pure RNA). The RNA yield; RNA integrity, as reflected by the distribution value 200; and RNA purity, as reflected by the 260/280 and the 260/230 nm absorbance ratios, were determined. The RNA was sequenced on the NovaSeq 6000 instrument using the TruSeq RNA Exome and SMARTer Stranded Total RNA-Seq Pico v3 library preparations kits. Our results highlight the impact of RNA extraction methodology on both preanalytical and sequencing-based gene expression results. Overall, methods B and C outperformed method A because these showed significantly higher fractions of uniquely mapped reads, an increased number of detectable genes, a lower fraction of duplicated reads, and better representation of the B-cell receptor repertoire. Differences among the extraction methods were generally more explicit for the total RNA sequencing method than for the exome-capture sequencing method. Importantly, the predicative value of quality metrics varies among extraction kits, and caution should be applied when comparing and interpreting results obtained using different methods.

Visual Signaling in the Semi-Fossorial Lizard Pholidobolus montium (Gymnophthalmidae).

It has been suggested that gymnophthalmids, like most semi-fossorial lacertoids, rely more in chemical cues to communicate, in comparison to other groups, like Iguanids, on which communication is mostly based on visual signaling. We present the first description of visual signaling in the Andean lizard Pholidobolusmontium (Gymnophthalmidae) and a complete ethogram based on ex situ observations (34 different types of behaviors including positions and simple movements). Through the design of conspecific stimulus experiments, we were able to recognize leg-waving as a visual signal, as it is only displayed in presence of conspecifics or in presence of a mirror and was one of first and most frequent displays in this context. We also detected other visual displays like neck-arching and tail-undulation which may also be relevant as visual signals. Based on our results, we propose that visual signaling is also possible in semi-fossorial lizards; however, further studies regarding chemical signal recognition and color detection are required to confirm our hypothesis.