Method: isolation of epithelial cell RNA from frozen jejunum segments while minimizing smooth muscle cell rNA contamination

Authors: YE, SHUMAO - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; MATTHAN, NIRUPA - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; LAMON-FAVA, STEFANIA - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; Solano-Aguilar, Gloria; TURNER, JERROLD - Brigham & Women'S Hospital; WANG, XIANG-DONG - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; WALKER, MAURA - Boston University; CHAI, ZHI - Pennsylvania State University; Lakshman, Sukla; Urban, Joseph; LICHTENSTEIN, ALICE - Jean Mayer Human Nutrition Research Center On Aging At Tufts University

Submitted to: Current Developments in Nutrition
Publication Type: Abstract Only
Publication Acceptance Date: 2/20/2020
Publication Date: 5/29/2020
Citation: Ye, S., Matthan, N.R., Lamon-Fava, S., Solano Aguilar, G., Turner, J.R., Wang, X., Walker, M.E., Chai, Z., Lakshman, S., Urban Jr, J.F., Lichtenstein, A.H. 2020. Method: isolation of epithelial cell RNA from frozen jejunum segments while minimizing smooth muscle cell rNA contamination. Current Developments in Nutrition. 4(Suppl_2):1182. https://doi.org/10.1093/cdn/nzaa056_029.
DOI: https://doi.org/10.1093/cdn/nzaa056_029

Interpretive Summary:

Technical Abstract: OBJECTIVE: High RNA quality is a prerequisite for accurate PCR and sequencing results. Dissecting a specific tissue fraction from frozen samples while maintaining RNA quality is challenging. Starting with frozen pig jejunum segments, we describe a novel method to isolate epithelial cell RNA while minimizing contamination with smooth muscle cell RNA. METHODS: Jejunum tissue segments from Ossabaw pigs (N = 30) were snap-frozen in liquid nitrogen upon harvest from a diet-statin study and stored at -80C. At the time of RNA isolation samples were incubated in prechilled RNAlater-ICE at -20C for 24 hours, opened longitudinally, and epithelium cleanly separated from the muscle layer using a scalpel and tweezers. Total RNA was extracted from the epithelium using TRI-reagent. RNA Quality Indicator (RQI) of total RNA was measured using Experion RNA StdSens Analysis kit. RNA-sequencing was performed on Illumina NextSeq 500 platform. Raw sequencing data were aligned to the domestic pig (sus scrofa 11.1) reference genome and applied to subsequent analyses. xCell, a gene signature-based tool trained by thousands of single cell types from various sources, was used to estimate enrichment of epithelial cells (target) and smooth muscle cells (contamination) across samples. A Student t-test was used to compare the enrichment scores of these two cell types between the current method and a traditional method (small jejunal pieces collected at necropsy and stored frozen until RNA isolation). RESULTS: RQI ranged from 8.6 to 9.7, above the standard for RNA sequencing (RQI > 8). The enrichment score of epithelial cells was significantly higher in the current method (mean = 0.030, SD = 0.006) compared to the traditional method (mean = 0.016, SD = 0.013) (p < 0.0001). The enrichment score of smooth muscle cells was significantly lower in the current method (mean = 0.043, SD = 0.035) compared to the traditional method (mean = 0.13, SD = 0.093) (p < 0.0001). CONCLUSIONS: The current method effectively maintained RNA quality and minimized contamination of epithelial with smooth muscle cells. This method may be applicable to other frozen archived tissues that require a single tissue source of RNA.