Name: RNA Isolation from Mouse Ocular Lens Epithelium and Fiber Cell Bulk Masses (Video) | JoVE Journal
Uploaded: 2025-10-10T12:45:00.000+00:00
Duration: 6 min 7 s
Description: Learn RNA isolation from mouse ocular lens epithelium and fiber cell bulk masses using microdissection and TRIzol extraction. This method enables transcriptomic analysis of distinct lens cell types for studying age-related pathologies.

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This work was funded by grant R01 EY032056 (to CC) from the National Eye Institute.

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</ol>

The authors have nothing to disclose.

The steps presented here demonstrate the ability to isolate RNA from separate lens compartments that can be successfully reverse transcribed into cDNA for molecular biology methods. Due to the sensitive nature of analysis methods, it is essential that the lens is clean of attached tissues at the dissection stage. This may require a few iterations of rolling the lens on a Kimwipe to achieve adequate cleanliness. Care must be taken to not penetrate the lens too deeply while separating the collagenous capsule from the fibro...

The ocular lens is a transparent organ that finely focuses light onto the retina to produce a clear image. The lens is comprised of two major cell types: a monolayer of epithelial cells covering the anterior hemisphere and fiber cells that make up the bulk mass of the tissue (Figure 1). The lens is enveloped by a collagenous basement membrane known as the lens capsule, to which epithelial cells are tightly adhered. As the lens grows, epithelial cells at the equator proliferate and differentiate into nascent shells of fiber cells that are layered onto the lens in a concentric manner1,2,3. Lifelong lens growth depends on the continuous proliferation of this small population of equatorial epithelial cells that make up the germinative zone4. As fiber cells mature, all cellular organelles are degraded to eliminate light scattering objects5,6,7,8,9,10,11 and maintain tissue transparency, and the innermost fiber cells are eventually compacted, resulting in a rigid lens center9,12. Due to the surrounding lens capsule, there is no cell turnover in the lens, and the seminal fibers remain at the center of the lens throughout life as new fibers are added at the tissue periphery or cortex. The fiber cells of the lens have different optical properties depending on their age and can provide a temporal snapshot of the varying biological characteristics at each given stage13.
Despite available surgical options, cataracts, defined as any opacity in the normally transparent lens, remain the leading cause of blindness in the world14. Cataracts can manifest in the lens epithelium, cortex, or nucleus with differing pathophysiologies15. However, the cellular and molecular mechanisms of cataract formation remain unclear16,17. To better understand how to prevent these different types of cataracts and develop alternatives to surgery, we must better understand how these different cell types maintain their homeostasis in the lens.
The epithelial and fiber cells play different physiological roles in the lens. Cell proliferation, for example, is restricted to the lens epithelium18. Meanwhile, the lens fibers comprise the bulk mass of the lens, providing structure and refractive properties to the lens9. To obtain a more nuanced perspective of the biological processes involved in the different compartments of the lens, epithelial and fiber cells must be investigated separately. Here, we present a method to isolate the epithelium from the fiber cell bulk mass, extract mRNA from each fraction, and analyze these transcripts using reverse transcription quantitative polymerase chain reaction (RT-qPCR).
<img alt="Eye lens structure diagram; light direction, longitudinal section, epithelial cells, lens fibers." class="xfigimg" src="/files/ftp_upload/69079/69079fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Lens anatomy diagram. The lens is composed of two cell types, a monolayer epithelial cells (blue and orange) covering the anterior hemisphere and a bulk mass of lens fibers (white). The tissue is surrounded by a thin collagenous membrane, known as the lens capsule (tan). Anterior epithelial cells (blue) are quiescent while equatorial epithelial cells (orange) proliferate, differentiate, and elongate to become new layers of fiber cells (white) at the lens periphery. New generations of fiber cells are overlaid onto previous generations of fibers in concentric shells. The oldest lens fiber cells are compacted into the center of the tissue. This figure has been modified from Cheng (2024)38. <a href="https://www.jove.com/files/ftp_upload/69079/69079fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Centrifuge and Rotor</td><td>Fisher Scientific</td><td>14285554PM</td><td></td></tr><tr><td>Chloroform, HPLC grade</td><td>Alfa Aesar</td><td>22920</td><td></td></tr><tr><td>Ethanol (190-proof)</td><td>Decon Labs</td><td>2801</td><td></td></tr><tr><td>Ethanol (200-proof)</td><td>Fisher BioReagents</td><td>BP2818</td><td></td></tr><tr><td>Fisherbrand Pellet Pestle</td><td>Fisher Scientific</td><td>12-141-363</td><td></td></tr><tr><td>Forceps, Curved; Student Tweezers #7</td><td>World Precision Instruments</td><td>501981</td><td></td></tr><tr><td>Forceps, Straight; Dumont #5</td><td>Fine Science Tools</td><td>11252-40</td><td></td></tr><tr><td>Forceps, Straight; Student Tweezers #4</td><td>World Precision Instruments</td><td>501978</td><td></td></tr><tr><td>Kimwipes, small</td><td>Kimberly-Clark</td><td>34155</td><td>Delicate task wipe</td></tr><tr><td>MicroAmp Adhesive Film Applicator</td><td>Applied Biosystems</td><td>4333183</td><td></td></tr><tr><td>MicroAmp EnduraPlate Optical 96-Well Fast Clear Reaction Plate with Barcode</td><td>Applied Biosystems</td><td>4483485</td><td></td></tr><tr><td>Micropipettors</td><td>Eppendorf</td><td>&amp;nbsp;01-671-120</td><td></td></tr><tr><td>Microscope, Dissection</td><td>Carl Zeiss</td><td>SteREO Discovery.V8</td><td></td></tr><tr><td>MiniAmp Optical Adhesive Cover</td><td>Applied Biosystems</td><td>4360954</td><td></td></tr><tr><td>MiniAmp Thermal Cycler</td><td>Applied Biosystems</td><td>A37834</td><td></td></tr><tr><td>MixMate Plate Vortex Mixer</td><td>Eppendorf</td><td>5353000529</td><td></td></tr><tr><td>Molecular Grade Water</td><td>Fisher Scientific</td><td>BP28191</td><td></td></tr><tr><td>Nanodrop One Microvolume UV-Vis Spectrophotometer</td><td>Thermo Scientific</td><td>ND-ONE-W</td><td></td></tr><tr><td>Nunc MicroWell MiniTrays</td><td>Fisher Scientific</td><td>12-565-154</td><td>Dissection Tray</td></tr><tr><td>PCR Tubes</td><td>VWR</td><td>201007-012</td><td></td></tr><tr><td>Petri Dish, 60 mm x 15 mm</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Phosphate Buffered Saline, 1x Dulbecco&#039;s</td><td>Gibco</td><td>14190-136</td><td></td></tr><tr><td>Pipette Tips, 10 &amp;micro;L Graduated</td><td>USA Scientific</td><td>1161-3700</td><td></td></tr><tr><td>Pipette Tips, 1250 &amp;micro;L XL Graduated</td><td>USA Scientific</td><td>1161-1720</td><td></td></tr><tr><td>Pipette Tips, 200 &amp;micro;L Profile Graduated</td><td>USA Scientific</td><td>1163-1700</td><td></td></tr><tr><td>QuantStudio 3 Real-Time PCR System</td><td>Applied Biosystems</td><td>A28567</td><td></td></tr><tr><td>Reverse Transcriptase, SuperScript IV VILO&amp;nbsp;</td><td>Invitrogen</td><td>11756050</td><td>Highly processive and thermostable reverse transcriptase</td></tr><tr><td>RNA Clean and Concentrator 5 Kit</td><td>Zymo</td><td>ZR1013</td><td></td></tr><tr><td>RNase-Free Water</td><td>Fisher BioReagents</td><td>BP2819</td><td></td></tr><tr><td>RNaseZap</td><td>Sigma</td><td>R2020</td><td>RNase decontamination solution</td></tr><tr><td>SealRite Microcentrifuge 1.5 mL Tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>SuperFine Vannas 8 cm</td><td>World Precision Instruments</td><td>501778</td><td>Scissors</td></tr><tr><td>TaqMan FAST Advanced Master Mix for qPCR</td><td>Applied Biosystems</td><td>4444557</td><td></td></tr><tr><td>TaqMan Gene Expression Assay (FAM) Probe (MTO)</td><td>ThermoFisher Scientific</td><td>4448892</td><td></td></tr><tr><td>TRIzol</td><td>Invitrogen</td><td>15596026</td><td>Acid-guanidinium-phenol solution</td></tr><tr><td>Vortex Mixer</td><td>Thermo Scientific</td><td>88880017</td><td></td></tr></tbody></table>

rna isolation from mouse ocular lens epithelium and fiber cell bulk masses

RNA Isolation from Mouse Ocular Lens Epithelium and Fiber Cell Bulk Masses

The lens is a specialized, transparent tissue in the anterior chamber of the eye that is comprised of two major cell types, epithelial cells and fiber cells. A monolayer of lens epithelial cells covers the anterior hemisphere of the lens, while most of the lens is composed of a mass of lens fiber cells. A collagenous basement membrane, known as the capsule, surrounds and encapsulates the entire tissue. Lens epithelial cells are strongly adhered to the lens capsule and can easily be separated from the bulk fiber mass by peeling the capsule away from the tissue. Difficulties obtaining sufficient RNA concentration from the low number of epithelial cells in the lens monolayer has previously impeded the study of epithelial cell vs. fiber cell transcriptomes. This protocol presents a method to cleanly separate and isolate the epithelial and fiber cell compartments and RNA concentration steps to allow subsequent transcriptomic experiments on epithelial cell samples from a single pair of mouse lenses. The ability to investigate the major cell types of the lens individually aids in investigating biological mechanisms of lens maintenance and dysregulation, enabling the characterization of cell-type-specific disruptions responsible for age-related lens pathologies.

Lens epithelium and fibers were isolated from 6- to 7-week-old wild-type mice. Three mice were used for these experiments, and 2 lens capsules or 2 fiber cell masses from each mouse were pooled together for one biological replicate. As described in the protocol, RNA was extracted using TRIzol reagent phase separation. On average, a pair of lens epithelium and fiber bulk masses yielded 0.8 µg (SD ± 0.2) and 9.7 µg (SD ± 2.3) of RNA, respectively. The average concentration in a 15 µL elution was 55.4 ng/µL (SD ± 16.3) and ...

This protocol details the separation of the mouse ocular lens epithelium and fiber cell bulk mass, followed by RNA isolation and qPCR analysis. This method allows for separating lens cell compartments for a more detailed analysis of the transcriptome and biological processes in epithelial cells vs. differentiated fiber cells.

The lens is a specialized, transparent tissue in the anterior chamber of the eye that is comprised of two major cell types, epithelial cells and fiber ...

Our research focuses on the lens, a specialized transparent tissue in the anterior chamber of the eye. The lens is composed of two cell types, epithelial cells and fiber cells that have different functions and diverse transcriptomes. There are difficulties in extracting a sufficient concentration of RNA from epithelial cells in the lens monolayer, which has impeded the study of transcriptomes in epithelial cell versus fiber cell.The ability to cleanly separate the major cell types of the lens allows deeper investigation of lens maintenance and dysregulation. This enables the characterization of cell type specific disruptions that lead to age-related lens pathologies. To begin, microdisect the lens from freshly enucleated eyes of a euthanized mouse.Gently roll the lens on a clean, delicate task wipe using curved forceps to remove any remaining adherent extra lenticular tissue. Using fine straight forceps, shallowly pierce the lens capsule near the equator, then gently peel the lens capsule away from the fiber bulk mass, leaving the lens epithelial cells attached to the capsule. Remove any large fiber pieces from the capsule that may have come off during decapsulation.Now gently grip the capsule with fine straight forceps and swirl it in PBS to dislodge any remaining fibers. Pipette 400 microliters of cold TRIzol reagent into a clean 1.5 milliliter micro centrifuge tube per sample. Deposit two lens capsules or two fiber bulk masses into each respective tubes.Tightly cap the tubes. Gently homogenize the lens fiber bulk masses in the tubes using a clean plastic pestle, then incubate. In the fume hood, add 200 microliters of chloroform per 400 microliters of TRIzol reagent into each tube.Close the tubes tightly and shake vigorously by hand for 15 seconds, keeping the thumb on the bottom of the tube and the forefinger on the cap. After incubating the samples at room temperature for 10 to 15 minutes to allow phase separation, centrifuge the samples at 14, 000 G for 15 minutes at four degrees Celsius. In the fume hood, carefully transfer the clear top aqueous phase into a clean 1.5 milliliter micro centrifuge tube and note the volume.Then add 200 proof ethanol to the aqueous phase in a one-to-one volumetric ratio. Now, gently mix the solution by inverting the tube several times. Transfer the mixed solution to an RNA spin column using a pipetter.Centrifuge the spin columns at 16, 000 G for 30 seconds at four degrees Celsius. Then discard the flow through and pipette 400 microliters of RNA prep buffer to the spin column. After centrifuging the columns again, discard the flow through.Add 700 microliters of RNA wash buffer to the spin column. Once the final wash is complete and the flow through has been discarded, centrifuge once more at 16, 000 G for 30 seconds at four degrees Celsius to ensure the spin column is dry. Move the dried spin column to a new, clean, and labeled 1.5 milliliter micro centrifuge tube.Then pipette 15 microliters of RNase-free water directly onto the membrane filter and incubate for two minutes at room temperature. Centrifuge the spin column one final time at 16, 000 G for 30 seconds at four degrees Celsius to elute the purified RNA. Now remove and discard the spin columns.The purified RNA will be present in the liquid collected in the micro centrifuge tubes. Incubate the RNA samples at 55 to 65 degrees Celsius for 10 minutes to promote resolubilization. Immediately place the samples on ice following the heating step.Store the samples at minus 80 degrees Celsius. Differential gene expressions were observed between the isolated epithelium and fiber cell bulk mass. The gene Gja1, also known as connexin 43, was expressed primarily in epithelial cells.In contrast, the gene Gja3, which encodes connexin 46, had higher expression in fiber cells. Gja8, or connexin 50, was expressed in both epithelial and fiber cells. Expression of Cdh1 encoding E-cadherin, was high in epithelial cells while expression of Cdh2, encoding N-cadherin, was detected in both epithelial cells and fiber cells.Pax6 expression was strongly enriched in epithelial cells and was nearly absent in fiber cells. In contrast, the Crygs gene encoding the gamma S crystallin protein exhibited highly elevated expression in fiber cells compared to epithelial cells.

rna-isolation-from-mouse-ocular-lens-epithelium-fiber-cell-bulk

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JoVE Journal

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Learn RNA isolation from mouse ocular lens epithelium and fiber cell bulk masses using microdissection and TRIzol extraction. This method enables transcriptomic analysis of distinct lens cell types for studying age-related pathologies.

RNA Isolation from Mouse Ocular Lens Epithelium and Fiber Cell Bulk Masses

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