Immunohistochemical analysis of placental tissues for <i>in vivo</i> evidence of DNA damage.

<p>Placental tissues were collected from women with preeclampsia [PE] [<b><i>A–D</i></b>] and gestational age-matched normotensive controls (n = 10 for each) [<b><i>E–H</i></b>]. Tissues were prepared and labeled with antibodies against γH2AX (a biomarker of DNA damage [<b><i>A,E</i></b>]), cytokeratin 7 (CK-7) (which stains trophoblast cells, see arrows [<b><i>B,F</i></b>]), and vimentin (which stains decidual cells, see arrow heads [<b><i>C,G</i></b>]) as described in the <i>Materials and Methods</i>. Negative controls [<b><i>D,H</i></b>] included saline in place of first antibody. Representative serial tissue sections are shown. γH2AX labeling (see arrow heads in <b><i>A</i></b>) was more intense in PE tissues than normotensive controls. The intensity of staining in decidua cells was quantified using the H-SCORE system (measured in relative staining intensity) and the data analyzed using GraphPad Prism software. Data are mean±SEM from a minimum of 3 separate experiments performed in triplicate; **<i>P</i><0.001[<b><i>I</i></b>].</p

DNA apurinic/apyrimidinic (AP) sites as a measure of DNA damage in cultured decidual and cytotrophoblast cells <i>in vitro</i>.

<p>Term decidual stromal cells (DSCs) and cytotrophoblast cells (CTs) were isolated, purified, and cultured with or without H₂O₂ 100 µM for 1 h as described in the <i>Materials and Methods</i>. The effect of H₂O₂ treatment on the number of DNA AP sites was estimated using a commercial ELISA-like assay that utilizes an aldehyde reactive probe (ARP) (Abcam, Cambridge, MA). Results are expressed as fold change in the number of DNA AP sites per 10⁵ nucleotides. **<i>P</i><0.001, ****<i>P</i><0.0001.</p

Western blot analysis.

<p>To investigate the effect of H₂O₂ treatment on γH2AX protein expression, Western blot analysis was performed on protein extracted from decidual stromal cells (DSCs) and cytotrophoblast cells (CTs) cultured with or without H₂O₂ 100 µM for 1 h as described in the <i>Materials and Methods</i>. Representative Western blots are shown, including α-tubulin control.</p

Comparison of two mechanical disaggregation methods of fresh lung tissues for extraction of high-quality RNA

Gene expression studies are widely used in medical, biological, and pharmaceutical research. Obtaining high-quality RNA from tissues is a prerequisite for high-quality data that should accurately represent gene expression levels in-vivo. The main source of technical bias, which could affect the results from transcriptomic studies, is variation in RNA quality. In this regard, tissue preparation is critical: different disruption techniques can affect RNA quality, influencing further applications. Mechanical disaggregation is a common, inexpensive, and simple method to obtain a high cell yield, demonstrated to efficiently disrupt the extracellular matrix and release single cells. However, its efficacy is operator-dependent, leading to poorly reproducible results. A fast, reproducible, and standardized technique could undoubtedly overcome this problem, avoiding wasting time and resources. In this study, our goal was to evaluate the impact of two mechanical tissue disruption techniques on the purity and quality of RNA extracted from fresh lung biopsies. The samples were processed in parallel using manual mechanical disaggregation or an automated mechanical device. The results showed that samples processed with the automated device had a higher integrity compared to those processed manually with a median Fragmentation Index of 0.86 and 0.71 respectively. This difference is statistically significant (p = 0.0084). Overall, our results indicated that the use of automatic mechanical disaggregation could undoubtedly help to overcome the technical biases related to fresh tissues processing.</p