# Optimized protocol to preserve RNA integrity for laser capture microdissection of bovine mammary epithelial cells

## TL;DR

Researchers developed a method to preserve RNA quality when isolating bovine mammary epithelial cells using laser capture microdissection for RNA sequencing.

## Contribution

An optimized LCM protocol was developed to maintain RNA integrity in bovine mammary epithelial cells for transcriptomic analysis.

## Key findings

- Staining with RNase inhibitor and xylene clearing improved RNA preservation.
- LCM dissection time under 15 minutes minimized RNA degradation.
- High-quality RNA suitable for RNA sequencing was successfully isolated from microdissected MEC.

## Abstract

Summary: A laser capture microdissection (LCM) protocol was developed for whole-transcriptome profiling of lactating bovine mammary epithelial cells (MEC). Mammary tissue after the biopsy was embedded in optimal cutting temperature (OCT) compound and frozen in liquid nitrogen vapor for long-term storage at -80°C. For the effect of fixation time, a staining solution with and without RNase inhibitor was optimized for a good morphology of the stained section with minimum impact on cellular RNA integrity. Mammary alveolar epithelial cells were microdissected for total RNA isolation. The bioanalyzer measurements on RNA isolated from pooled microdissected MEC showed good-quality RNA suitable for RNA sequencing. The results of LCM sequencing revealed differentially expressed genes and transcriptomic signatures of bovine MEC.

Summary: A laser capture microdissection (LCM) protocol was developed for whole-transcriptome profiling of lactating bovine mammary epithelial cells (MEC). Mammary tissue after the biopsy was embedded in optimal cutting temperature (OCT) compound and frozen in liquid nitrogen vapor for long-term storage at -80°C. For the effect of fixation time, a staining solution with and without RNase inhibitor was optimized for a good morphology of the stained section with minimum impact on cellular RNA integrity. Mammary alveolar epithelial cells were microdissected for total RNA isolation. The bioanalyzer measurements on RNA isolated from pooled microdissected MEC showed good-quality RNA suitable for RNA sequencing. The results of LCM sequencing revealed differentially expressed genes and transcriptomic signatures of bovine MEC.

•We optimized staining and dehydration steps to minimize RNA degradation in stained tissue within 5 minutes.•Precise LCM within 15 minutes further prevented RNA degradation.•High-quality RNA isolated from MEC was suitable for RNA sequencing.

We optimized staining and dehydration steps to minimize RNA degradation in stained tissue within 5 minutes.

Precise LCM within 15 minutes further prevented RNA degradation.

High-quality RNA isolated from MEC was suitable for RNA sequencing.

Laser capture microdissection (LCM) is a popular technique for isolating specific cell types from tissues for medical research. However, the LCM procedure for isolating bovine mammary epithelial cells (MEC) for high-throughput transcriptome profiling has been lacking. The quality and quantity of RNA in LCM samples can be significantly affected by tissue treatment, time for dissection, and the total area of cells dissected. The objective of this study was to optimize the procedures for isolating bovine MEC from mammary tissues for consistent isolation of intact RNA suitable for downstream high-throughput RNA sequencing. Mammary tissues were biopsied (approximately 10–15 min per biopsy) from lactating cows, stored at −80°C, cryosectioned, and processed for LCM. Using LCM software, closed lines were selected and drawn around MEC visible within the alveoli of stained sections and microdissected. Degradation of MEC RNA was minimized by minimal exposure of tissue to aqueous media, the addition of RNase inhibitors in staining solution, and reducing LCM dissection time to less than 15 min (13.6 ± 0.52 min; mean ± SEM). Results showed that tissue fixation with chilled 70% ethanol, histone staining (with RNase inhibitor), dehydration in absolute ethanol, and final clearing in xylene preserved the RNA quality compared with staining sections without RNase inhibitor, no xylene clearing, and longer microdissection times. Using this approach, high-quality RNA was successfully obtained for RNA sequencing.