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An optimized laser capture microdissection protocol for intact RNA isolation from lipopolysaccharide treated mammary epithelial cells.

R. K. Choudhary

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06-22-2020

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Abstract:

M87
An optimized laser capture microdissection protocol for intact RNA isolation from lipopolysaccharide treated mammary epithelial cells.
R. K. Choudhary*1, T. B. McFadden2, E. M. Shangraw2, R. O. Rodrigues2, F.-Q. Zhao1. 1Department of Animal and Veterinary Sciences, University of Vermont Burlington, VT, 2Division of Animal Sciences, University of Missouri Columbia, MO.

Laser capture microdissection (LCM) is one popular technique for isolating specific cell types from tissues. However, RNA quality, quantity and integrity in LCM samples can be greatly affected by tissue treatment, length of dissection and the total areas of cells dissected. In this study, we optimized methodology to obtain high quality RNA from mammary epithelial cells collected from bovine mammary glands treated with lipopolysaccharide (LPS) and determined if LPS affected the quality, quantity and integrity of RNA. Ten multiparous cows were used. Five treatment (T) cows received one intramammary dose of LPS (50 �g in 10 mL saline; TL) in each of 2 ipsilateral glands while the contralateral glands received saline (10 mL; TS). Likewise, in 5 control (C) cows, saline (CS) was infused into 2 ipsilateral glands and the other glands remained uninfused (CU). Mammary tissues were collected at 0, 3 and 12 h, relative to infusions and processed for LCM. After staining, tissue sections were visualized using an epifluorescence microscope with attached computer and manually selected areas of epithelial cells were dissected using LCM. Time of dissection was kept minimal (13.6 + 0.52 min; mean + SE) to avoid RNA degradation, and areas of dissected cells were consistent across treatment groups and times. Results showed that fixation of tissue sections with chilled 70% ethanol, histogene staining (with RNase inhibitor), dehydration in absolute ethanol, and final clearing in xylene was able to preserve quality of RNA isolated from microdissected cells. Analysis of total RNA from mammary epithelial cells harvested by LCM showed RNA yield per unit area was affected by treatment, time and interaction of treatment x time, suggesting that LPS increased transcription or reduced RNA degradation in epithelial cells. In addition, RNA integrity number (RIN) was affected by treatment and time x treatment interaction (P ≤ 0.01). In summary, we developed an optimized LCM protocol to reproducibly obtain high-quality RNA and suggest that LPS treatment may affect RNA yield of mammary epithelial cells.

Keywords: mastitis, RNA quality, transcription.