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 manipulating other angiogenesis-associated genes in animals.

We have shown that angiogenic PKD-1 signaling and the transcription of angiogenic gene CD36 are critical for endothelial cell (EC) differentiation and functional angiogenesis^3,4,5,6. To determine molecular mechanisms of angiogenic and metabolic signaling in gene transcription and EC transdifferentiation, we have created genetically engineered TRAP mice with specifically deleted angiogenic genes on the basis of TRAP technique^1,2. Furthermore, in our TRAP animals, not only do they have pkd-1 or cd36 gene deficiency in the vascular endothelia or global deletion of cd36 gene, but an enhanced green fluorescence protein (EGFP) is also genetically tagged onto EC's translating ribosomes. TRAP permits affinity purification of ribosome-bound mRNA directly from the vascular endothelia of targeted tissues, enabling the analysis of gene expression and identification of new transcriptomes that are associated with EC differentiation and angiogenesis directly under in vivo conditions. We have successfully isolated ribosome-bound RNA from the endothelia in these genetically engineered animals. The purified RNA can be used for further characterization of angiogenic or arteriogenic genes in the regulation of EC differentiation and functions. This protocol provides a step-by-step guide to implement the TRAP approach for the isolation of mRNA in ECs directly in vivo.

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 inhibitors (one mini tablet per 10 mL) and RNase inhibitor (10 µL/mL).
   2. Store in a 4 °C fridge for up to 1 month.
2. Prepare a high-salt polysome wash buffer to concentrations of 10 mM HEPES, pH 7.4, 350 mM KCl, 5 mM MgCl₂, 1% vol/vol CA-630, 0.5 mM DTT, and 100 µg/mL cycloheximide.
   1. Add following reagents to 500 mL of RNase-free deionized water: 1.19 g of HEPES, 13.05 g of KCl, 0.24 g of MgCl₂, 5 mL of nonionic, non-denaturing detergent, 5 of 7.7 mg tubes of DTT, and 0.5 mL of cycloheximide, and NaOH as needed until pH 7.4.
   2. Store in 4 °C fridge for up to 1 month.
3. Bind anti-GFP antibody to Protein G magnetic beads prior to starting experiment.
   1. Add 10 µg of anti-GFP antibody diluted in 200 µL of PBS to Protein G beads.
   2. Incubate with end over end rotation for 10 minutes at room temperature.
   3. Place the beads on a magnetic rack and remove the supernatant.
   4. Suspend the beads in 200 µL of PBS and store in 4 °C fridge for up to 1 week.
4. Prepare ice-cold PBS with 100 µg/mL cycloheximide.
   1. Add 1 volume of cycloheximide solution (100 mg/mL) to 999 volumes of ice-cold PBS.

2. Isolate and lyse desired tissues

1. Euthanize mice by IP injection of ketamine (500 mg/kg/body weight) and xylazine (10 mg/kg/body weight) and isolate desired tissues (i.e., heart, lung). Immediately proceed to next step.
2. Place desired tissues into 500 µL of ice-cold PBS with 100 µg/mL cycloheximide.
3. Mince tissue into a cell suspension with a motor-driven homogenizer or a small-clearance glass homogenizer. If using a motor-driven homogenizer, limit homogenization to less than 1 minute at low frequency (<15,000 Hz) to avoid RNA denaturation.
4. Suspend cell pellet in 200 µL of lysis buffer by pipetting and redrawing up buffer several times. Further homogenize cell suspension with 10 strokes in a small-clearance glass homogenizer or for 15 seconds at low frequency (<15,000 Hz) in a motor driven homogenizer.
5. Centrifuge homogenates for 10 min at 2,000 x g at 4 °C to pellet nuclei and large cell debris, and keep the supernatant.
6. Add nonionic, non-denaturing detergent to 1% vol/vol and DHPC to 30 mM to the supernatant. Incubate on ice for 5 min.
7. Centrifuge lysate for 10 min at 16,000 x g to pellet insoluble material. Transfer and keep 15% of clear lysate as input for future steps.

3. Isolate ribosome/mRNA complexes

1. Add 50 µL of antibody-bound beads to cell-lysate supernatant and incubate mixture at 4 °C with end-over-end rotation for 30 min. This is where the anti-GFP antibodies will bind the GFP-tagged ribosomes, allowing us to further isolate the RNA from these ribosomes.
2. Collect beads on a magnetic rack and wash 5 times with high-salt polysome wash buffer.
   1. Draw up and discard liquid once beads have collected on the side of the tube. Then pipette and redraw up 200 µL of high-salt polysome wash buffer several times. Repeat this step 5 times and discard all buffer following final repetition. Immediately proceed to next step.

4. Isolate mRNA

1. Place beads in RLT buffer. The following steps are taken directly from the RNeasy mini kit protocol and were not expanded on in any way.
   CAUTION: RLT buffer contains guanidine salts; do NOT mix with bleach.
2. Centrifuge lysate for 3 min at full speed 13,000 rpm or 16,000 g at 4 °C. Carefully remove supernatant of 350 µL by pipetting and transfer it to a new microfuge tube. Use only this supernatant (lysate) in subsequent steps.
3. Add an equal volume of 70% ethanol into the microfuge tube.
4. Transfer up to 700 µL of the sample, including any precipitate that may have formed, to a spin column placed in a 2 mL collection tube. Close the lid gently and centrifuge for 15 s at ≥8,000 x g to wash the spin column membrane. Discard the flow-through.
5. Add 350 µL of buffer RW1 to the spin column. Close the lid gently and centrifuge for 15 s at ≥8,000 x g to wash the spin column membrane. Discard the flow-through and reuse the collection tube in next step.
   CAUTION: Buffer RW1 contains guanidine salts; do NOT mix with bleach.
6. Add 350 µL of buffer RW1 to the spin column. Close the lid gently and centrifuge for 15 s at ≥8,000 x g. Discard the flow-through.
7. Add 500 µL of buffer RPE to the spin column. Close the lid gently and centrifuge for 15 s at ≥8,000 x g to wash the spin column membrane. Discard the flow-through.
8. Add 500 µL of buffer RPE to the spin column. Close the lid gently and centrifuge for 2 min at ≥8,000 x g to wash the spin column membrane. Then carefully remove the spin column from the collection tube, ensuring that the column does not contact the flow-through.
9. Place the spin column in a new 2 mL collection tube and discard the old tube with the flow-through. Close the lid gently and centrifuge at full speed for 1 min to remove residual buffer.
10. Place the spin column in a new 1.5 mL collection tube. Add 30-50 µL of RNase-free water directly to the spin column membrane. Close the lid gently and centrifuge for 1 min at ≥8,000 x g to elute the RNA.
11. If expected RNA yield is >30 µg, repeat step 4.10 with another 30-50 µL of RNase-free water, or using elute from Step 4.10 (if high [RNA] is required). Reuse collection tube from Step 4.10.
12. Use purified RNA for downstream analysis including RNA-sequencing or real time quantitative PCR or store RNA dissolved in RNase-free H₂O at -80 °C for up to 1 year.

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