Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Summary

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Abstract

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision ¹. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

Introduction

The main therapeutic objective in retinal detachment (RD) is to find a way to limit photoreceptor cell damage and retinal inflammation resulting from the separation of the photoreceptors from the retinal pigmented epithelial cells. During RD, RPE cells are activated, migrate, dedifferentiate, and proliferate at the surface of the detached retina, exerting contractile forces leading to complications. Transcriptomics analysis of RD is a way to identify target genes with modified expression following RD and hence future therapeutic molecules that could improve final visual outcome in combination with surgery. It is well known ribonucleic acid (RNA) is not stable as is de....

Protocol

1. jouRNAl: Procedure to Recover the Specimens from the Surgical Block

In the lab

1. Get an importation contract from express shipping company.
2. Fill 10 (or 25) shipping forms with the postal address of the laboratory indicating the contact person in the laboratory (Phone number and Email address).
3. Prepare 10 (or 25) samples forms numbered 1 to 10 (or 25). These forms include dedicated spaces to inform on a) anonymous identification of the patient, b) the date of the surgery and c) any additional remarks the surgeon would like to add. Prepare 10 (or 25) padded envelopes (150 x 210 mm).
4. Prepared using RNAse....

Results

The procedure jouRNAl (Figure 1) allows us recover specimens of retina from the surgical block to purified RNA, and to analyze the transcriptome of retinal detachment. Retinal detachment results in the upregulation of the monocyte chemotactic MCP1 gene CCL2, and in the decrease in expression of the rod photoreceptor transducing gene GNAT1, the short wave cone opsin OPN1SW, and the homeogene CRX (Figure 2). The induction of CCL2 is resultin.......

Discussion

The development of a procedure for tissue recovery from the surgical block has been essential to the transcriptome analysis of retinal detachment. One should notice that this type of surgery is practiced in emergency and that the ophthalmologists operating have little time to participate in a biological research program when they operate. This retinectomy is also performed stochastically in each service, so that the easier way to reach statistical numbers is to work with a network. In such network, the standardiza.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Centrifuge	Beckman Coulter	Aventi J-E
Rotor	Beckman Coulter	JS-5.3
Ultracentrifuge	Beckman Coulter	LE 80K
Rotor	Beckman Coulter	SW41
Power supply	Biorad	Pac 3000
PolytronTM	Kinematica	PT 2100	Supplied with PT-DA 2105:2
Agarose gel electrophoresis device	Biorad	MiniGel Cell GT
Imaging System	Biorad	GelDoc-It Imager
5 ml sterile polyethylene tube	Greiner	115261
Sterile polyallomer centrifuge tube	Beckman Coulter	331372
Chemical reagents	Sigma		Molecular biology grade (RNase and DNase free products)
Cleaning Solution	VWR	RBS
Microarray chip	Affymetrix	Human U133 plus 2 array