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• W3032103175 abstract "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. Jejunum tissue segments from Ossabaw pigs (N = 30) were snap-frozen in liquid nitrogen upon harvest from a diet-statin study and stored at −80°C. At the time of RNA isolation samples were incubated in prechilled RNAlater-ICE at −20°C 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). 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). 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. USDA-ARS-NEA, JM-USDA-HNRCA, and Tufts University." @default.
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• W3032103175 date "2020-06-01" @default.
• W3032103175 modified "2023-10-02" @default.
• W3032103175 title "Method: Isolation of Epithelial Cell RNA from Frozen Jejunum Segments While Minimizing Smooth Muscle Cell RNA Contamination" @default.
• W3032103175 doi "https://doi.org/10.1093/cdn/nzaa056_029" @default.
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