# Comparison Between Two Methodologies of Sample Preservation for RNA Extraction in Naturally Delivered Ovine Placenta

## TL;DR

This study shows that high-quality RNA can be extracted from naturally delivered sheep placenta when preserved using snap freezing, but not with RNAlater®.

## Contribution

The study introduces a non-invasive method for RNA extraction from naturally delivered ovine placenta using snap freezing.

## Key findings

- Snap freezing preserved RNA quality better than RNAlater® in naturally delivered ovine placenta.
- RNA concentration was higher in RNAlater®-preserved samples, but RNA quality was significantly lower.
- Timing of placental delivery did not affect RNA quantity or quality.

## Abstract

The study of the placenta is relevant due to its central role in reproduction. Evaluating gene expression is a valuable tool to assess placental function. To accomplish this, tissue samples are used to extract RNA, a molecule whose quality is essential for successful gene expression studies. A challenge in small ruminants, such as sheep, is that samples are collected postmortem (non-recovery) or surgically (invasive). An alternative could be to use naturally delivered placenta. However, the placenta is rich in enzymes that degrade RNA, being unknown if postpartum sheep placenta will be suitable for RNA extraction. We evaluated if high-quality RNA could be obtained from delivered ovine placenta and compared two preservation methods: 1. snap frozen (SF, samples preserved using liquid nitrogen) and 2. RNAlater® (LTR, samples submerged in RNA preserving solution). Placental expulsion timing has great variety among sheep, so we evaluated if this has an impact on RNA quantity and quality. Our major findings indicate that it is possible to extract RNA from naturally delivered placenta, but RNA quality was acceptable only when using the SF method. We also found that the timing of placental delivery did not affect the quality of RNA.

Placental samples for RNA extraction are collected via non-recovery (euthanasia) or invasive (surgery) methods in small ruminants, such as sheep. Alternatively, delivered placentas could be used, but the feasibility of obtaining high-quality RNA from this tissue is unknown in sheep. We aimed to evaluate the possibility of extracting RNA from naturally delivered ovine placenta, comparing two preservation methods. Twenty-seven single-pregnant sheep were monitored 24/7 from gestational day 140 to parturition. Tissue was collected after placental delivery, preserved using snap frozen (SF, n = 27) and RNAlater® (LTR, n = 27) techniques, and processed for RNA extraction using a commercial kit. RNA concentration (ng/µL), A260/280, and RNA quality number (RQN) were measured. Concentration was higher (p < 0.001) in LTR (70.39 ± 6.3) than in SF (49.77 ± 10.5), A260/280 was higher (p = 0.045) in SF (2.06 ± 0.01) than in LTR (2.03 ± 0.01), and RQN was higher (p < 0.0001) in SF (6.81 ± 0.24) than in LTR (2.84 ± 0.24) samples. Timing of placental delivery did not affect the evaluated indicators. Results indicate that extracting high-quality RNA from delivered placentas preserved via the snap-frozen technique is possible, supporting a method that aligns with the refinement principle of animals used in research.