Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Summary

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Abstract

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Introduction

In complex tissues such as the mammalian brain, heart and lung, the high levels of cellular heterogeneity complicate the analysis of gene expression data derived from whole tissue samples. To observe gene expression profiles in a particular cell type in vivo, a new methodology has been developed recently, which allows the interrogation of the entire translated mRNA complement of any genetically defined cell type. This methodology is known as the translating ribosome affinity purification (TRAP) technique^1,2. It is a useful tool to study endothelial cell biology and angiogenesis when combined with genetically m....

Protocol

For animal experiments, all methods described here have been approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

1. Prepare reagents

1. Prepare lysis buffer to concentrations of 10 mM HEPES, pH 7.4, 150 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, 100 µg/mL cycloheximide, protease inhibitors, and recombinant RNase inhibitors to concentrations as described below.
   1. Add following reagents to 500 mL of RNase-free deionized water: 1.19 g of HEPES, 5.59 g of KCl, 0.24 g of MgCl₂, 35 mg of DTT, 0.5 mL of cycloheximide, and NaOH as needed until pH 7.4, EDTA-free protease....

Results

Our previous studies^4,7 suggest that CD36 may function as a switch for arteriolar differentiation and capillary arterialization via the LPA/PKD-1 signaling pathway. To study whether the LPA/PKD-1-CD36 signaling axis is essential for arteriogenesis in vivo, we have established the novel TRAP lines that not only have global cd36 deficiency or endothelial-specific-cd36- or pkd-1-deficiency but also permit selective isolation of ribosome-bound RNA from cre-marked cel.......

Discussion

Angiogenesis is a complex multistep process, in which EC-specific angiogenic gene transcription and expression play an essential role in EC differentiation and angiogenic reprogramming^3,4. To overcome the barriers from the cellular diversity and architectural complexity for better understanding the function of the mammalian vascular system at a molecular level in vivo, we have created EC-specific TRAP mice, accompanied by EC-specific cd36, EC-specific

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2100 Electrophoresis Bioanalyzer with Nanochips and Picochips	Agilent	G2939AA, 5067-1511 & 5067-1513
Cell scrapers	Sarstedt	83.1832
Homogenizers	Fisher Scientific	K8855100020
Magnet (Dynamag-2)	Invitrogen	123-21D	Will depend on purification scale; samples in 1.5-mL tubes can be concentrated on a DynaMag-2
Minicentrifuge	Fisher Scientific	05-090-100
NanoDrop 2000C spectrophotometer	Thermo Scientific	ND-2000C
Refrigerated centrifuge	Eppendorf	5430R	with rotor for 1.5-mL microcentrifuge tubes
RNase-free 1.5mL microcentrifuge tubes	Applied Biosystems	AM12450
Rnase-free 50-mL conical tubes	Applied Biosystems	AM12501
RNase-free 1000-μl filter tips	Rainin	RT-1000F
RNase-free 200-μl filter tips	Rainin	RT-200F
RNase-free 20-μl filter tips	Rainin	RT-20F
Rotor for homogenizers	Yamato	LT-400D
Tube rotator, Labquake brand	Thermo Fisher	13-687-12Q