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Method Article

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

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DOI:

10.3791/69079

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October 10th, 2025

 ,  , 
1School of Optometry and Vision Science Program, Indiana University

In This Article

Summary

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.

Abstract

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.

Introduction

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).

Eye lens structure diagram; light direction, longitudinal section, epithelial cells, lens fibers.
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. Please click here to view a larger version of this figure.

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Protocol

All animal experiments were conducted in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" and an approved protocol from the Institutional Animal Care and Use Committee (IACUC) of Indiana University.

1. Work area, equipment, and reagent preparation

  1. Autoclave pipette tips before use and designate boxes of autoclaved tips for RNA experiments to avoid RNase contamination.
  2. Treat the work surface area in the fume hood and tissue homogenization pestles with RNase decontamination solution, thoroughly rinse with deionized water, and dry.
  3. Soak dissection tools (curved forceps, straight forceps, and microdissection scissors) in 70% ethanol for 5 min and dry with a delicate task wipe before use.
  4. Use brand-new dissection trays and Petri dishes for dissection and tissue isolation steps.
  5. Aliquot 400 µL of cold TRIzol acid-guanidinium-phenol reagent into DNAse- and RNAse-free 1.5 mL microcentrifuge tubes in the fume hood.

2. Mouse lens dissection

  1. Euthanize adult mice using CO2 overdose followed by cervical dislocation as a secondary measure.
  2. Enucleate the eyes using curved forceps.
    1. Gently depress the tissue surrounding the eye with the forceps, causing the eye to protrude from the socket. Close the forceps underneath the eye and remove by pulling steadily upwards.
    2. Transfer the eyes into a 1.5 mL microcentrifuge tube with 750-1000 µL of 1x phosphate buffered saline (PBS).
  3. For dissection, transfer the eyes to wells in a dissection tray with fresh 1x PBS.
  4. Under a dissection microscope, use straight forceps to hold the base of the optic nerve and cut the nerve as close to the eye as possible with sharp microdissection scissors.
  5. Carefully insert fine straight forceps into the opening where the optic nerve had been connected, and pinch to hold the tissue in place.
  6. Insert the tip of the microdissection scissors into the same opening and carefully cut from the posterior pole of the eye towards the corneal-scleral junction. Do not insert the instruments too deeply to avoid damaging the lens.
    NOTE: The rodent lens occupies a large volume in the eye, and thus, shallow cuts are recommended to prevent puncturing the lens.
  7. Cut along the corneal-scleral junction using the microdissection scissors, separating at least half of the circumference of the eye.
  8. Use the straight forceps to gently invert the eye tissues while pushing on the cornea, extruding the lens through the corneal-scleral incision.
  9. Carefully remove any large, attached tissues from the lens using fine straight forceps.
  10. If needed, gently roll the lens on a clean, delicate task wipe using curved forceps to remove any remaining adherent extralenticular tissue.
    NOTE: Roll the tissue quickly across the task wipe, and do not allow excessive drying of the tissue. There may be small opacities on the lens surface due to dry spots on the lens capsule. These opacities are generally reversible when the lens is placed back into 1x PBS and will not affect subsequent steps.
  11. Transfer the cleaned lens into a 6 cm Petri dish with 1x PBS.
  12. Using fine straight forceps, shallowly pierce the lens capsule near the equator, and gently peel the lens capsule away from the fiber bulk mass. The lens epithelial cells will remain attached to the capsule. Gently remove any large fiber pieces from the capsule that may have come off with decapsulation.
  13. Gently grip the capsule with fine straight forceps and swirl in the 1x PBS to remove any remaining fibers. Any loosely attached fiber cells will dissociate from the capsule and epithelial cells.

3. RNA isolation

NOTE: A summarized workflow for RNA isolation is shown in Figure 2.

  1. Deposit 2 lens capsules or 2 fiber bulk masses into respective 1.5 mL microcentrifuge tubes containing 400 µL of cold TRIzol reagent.
    NOTE: Fiber masses should be moved from the dish to the tube using curved forceps to scoop up the tissue.
  2. Tightly cap and move the tubes to a chemical fume hood for subsequent steps.
  3. Gently homogenize the lens fiber bulk masses using a clean pellet pestle.
    NOTE: Be sure to break up the fiber mass completely.
  4. Incubate samples (lens capsules or homogenized fiber bulk masses) in TRIzol for 30 min at room temperature in the fume hood.
  5. In the fume hood, add 200 µL of chloroform per 400 µL of TRIzol reagent to each tube. Close the tubes tightly and shake vigorously by hand for 15 s, keeping the thumb on the bottom of the tube and the forefinger on the cap.
    NOTE: The 1.5 mL microcentrifuge tubes noted in the materials list are used due to the tight seal. If using other brands of microcentrifuge tubes, test the tube closure and seal before shaking, as TRIzol and chloroform solution can leak from under the cap of tubes that do not have a tight seal.
  6. Incubate samples for 10-15 min at room temperature to allow for phase separation.
    NOTE: There should be a pink TRIzol reagent layer at the bottom of the tube containing lipids and protein, a white layer between phases containing DNA, and a clear aqueous phase at the top containing RNA. The white DNA layer may be more difficult to see in the epithelial cell samples.
  7. Centrifuge samples at 14,000 x g for 15 min at 4 °C.
    NOTE: Carefully move tubes into and out of the centrifuge, taking care not to disturb the phases in transfer.
  8. Assemble tubes and reagents from a RNA Clean and Concentrator kit. Spin column tubes are fitted into a collection tube from the kit.
  9. In the fume hood, carefully transfer the clear aqueous phase (the top layer) into a clean 1.5 mL microcentrifuge tube, taking note of the volume.
    NOTE: Avoid disturbing, touching, or drawing up the white DNA layer beneath the aqueous phase. Tilt the tube when transferring the aqueous phase to avoid disturbing the white layer. It is preferable to leave a small amount of the aqueous phase in the tube rather than contaminate the sample. Typically, for 200 µL of added chloroform, 180-190 µL of aqueous phase can be recovered.
  10. Add 200-proof ethanol to the aqueous phase in a 1:1 volumetric ratio.
    NOTE: RNA Binding Buffer from the concentrator kit can be added in a 2:1 volumetric ratio to the aqueous phase prior to adding ethanol. This step is skipped to increase RNA yield and because the RNA has sufficient purity after isolation.
  11. Gently mix by inverting the tube several times or by pipetting the solution up and down. Transfer the mixed solution to a RNA spin column using a pipettor.
    NOTE: Be careful not to touch the filter with the pipette tip.
  12. Centrifuge spin columns at 16,000 x g for 30 s at 4 °C.
    NOTE: It is recommended to label the lid of the column and the side of the collection tube to keep the sets of tubes organized and in case the cap breaks off in the subsequent centrifugation step.
  13. Discard the flowthrough and add 400 µL of RNA Prep Buffer to the spin column.
  14. Centrifuge spin columns at 16,000 x g for 30 s at 4 °C.
  15. Discard the flowthrough and add 700 µL of RNA Wash Buffer to the spin column.
  16. Centrifuge spin columns at 16,000 x g for 30 s at 4 °C.
  17. Discard the flowthrough and add 400 µL of RNA Wash Buffer to the spin column.
  18. Centrifuge spin columns at 16,000 x g for 1 min at 4 °C.
  19. Discard the flowthrough and centrifuge once more at 16,000 x g for 30 s at 4°C to ensure the spin column is dry.
  20. Move the spin column to a new labeled 1.5 mL microcentrifuge tube.
  21. Add 15 µL of RNase-free water onto the membrane filter. Incubate for 2 min at room temperature.
    NOTE: Be careful not to touch the filter with the pipette tip.
  22. Centrifuge the spin column at 16,000 x g for 30 s at 4 °C to elute the purified RNA.
    NOTE: The cap of the 1.5 mL microcentrifuge tube will remain open during centrifugation. Carefully place the spin columns and microcentrifuge tubes such that the open caps rest against the rotor lid so it does not swing and break during centrifugation. The lids should trail the direction of the rotor rotation.
  23. Remove and discard the spin columns. The RNA will be in the liquid collected in the 1.5 mL microcentrifuge tubes.
  24. Tightly close the 1.5 mL microcentrifuge tubes and incubate RNA samples at 55-65 °C for 10 min to promote resolubilization.
  25. Immediately place samples on ice after the heating step.
  26. Store samples at -80 °C or reverse transcribe into cDNA for long-term storage.
    NOTE: RNA samples were stored for up to 3 months with no noticeable concentration loss. Aliquot RNA samples to smaller volumes for storage to minimize freeze-thaw cycles and prevent degradation.

4. RNA concentration measurement using a UV-Vis Spectrophotometer

  1. Keep the RNA samples on ice, power on the NanoDrop (UV-Vis Spectrophotometer), and allow the instrument to initialize.
  2. Under the Nucleic Acids menu, select the RNA quantitation module.
  3. Blank the UV-Vis Spectrophotometer by pipetting 2.0 µL of eluent from the RNA concentration step onto the pedestal, in this case, molecular-grade water. Lower the instrument arm and read the blank sample.
  4. Lift the instrument arm, clean the sensor pedestal using a clean, dry delicate task wipe, and followed by another wipe wetted with deionized water, followed by a dry wipe once again.
  5. If desired, enter sample details into the UV-Vis spectrophotometer interface.
  6. Load 2.0 µL of the RNA sample onto the pedestal, lower the arm, and quantitate.
  7. Repeat steps 4.4 through 4.6 to clean the pedestal between samples.
  8. When finished, save and export the experiment for further quantification if necessary.

5. Reverse transcription

  1. Using a highly processive and thermostable reverse transcriptase, reverse transcribe 2.0 µg of lens fiber RNA and all the extracted epithelial RNA using the manufacturer's recommended protocol. Mix reagents and RNA for each sample in a clean PCR tube. Keep all solutions and RNA samples on ice.
    NOTE: This transcriptase can reverse transcribe up to 2.5 µg of RNA per reaction. A full conversion from RNA to cDNA is assumed, so the RNA starting concentration is used as the presumptive cDNA concentration.
  2. Run the reaction in the thermal cycler using the conditions listed in Table 1.
  3. Store reverse-transcribed cDNA at -80 °C or proceed to the qPCR step.
    NOTE: cDNA samples were stored for up to 4 months with no noticeable degradation, as cDNA is more stable than RNA. Samples can likely be stored for longer, provided freeze-thaw cycles are minimized.
StepTemperature (°C)Time (min)Cycles
Annealing25101
Reverse Transcription50101
Enzyme Inactivation8551
Hold4∞1

Table 1: Thermocycler conditions for reverse transcription. These conditions are attuned to a specific, highly processive and thermostable reverse transcriptase. Run conditions may vary depending on the enzyme and kit used.

6. Quantitative real-time PCR

  1. Prior to starting reactions, prepare cDNA stock solutions to the desired concentration by diluting with molecular-grade water. In these experiments, 1 µL of cDNA will be used at 5 ng/µL for 5 ng/well reactions. Keep all solutions and cDNA samples on ice.
    NOTE: TaqMan array plates (0.1 mL format) are recommended to accommodate reactions of 5-50 ng cDNA per well. Making aliquots of cDNA is useful to limit freeze-thaw cycles. In this study, samples were limited to a maximum of 5 freeze-thaw cycles.
  2. Prepare a working master mix comprised of Advanced Master Mix and probes to commercial recommendations. See section 7 for an example preparation. Keep all solutions and cDNA samples on ice.
    NOTE: A pair of probes with two different dyes is commonly used per well. For example, in this study, Crygs-FAM-MGB was used as the gene of interest probe, and Ppia-VIC-MGB as an internal control. Commercial recommendations for final probe and primer concentrations are 250 nM and 900 nM, respectively. If a target is very highly expressed, a primer-limited probe may be needed to prevent depletion of reaction reagents.
  3. Load the cDNA stock (1 µL) into appropriate wells on the qPCR plate, followed by the working master mix (9 µL). Mix solutions by pipetting up and down slowly while adding the master mix. Ensure that the drop of cDNA is mixed into the solution and avoid bubbles.
    NOTE: Depending on the sensitivity of the target, the plate may need to be loaded on ice.
  4. Seal the qPCR plate by carefully applying an optical adhesive cover. Use an adhesive film applicator to smooth the cover and ensure a tight seal around each well.
  5. Vortex plates at 1000 RPM for 10 s to mix.
  6. Centrifuge plates at 1000 x g for 2 min at room temperature to ensure there are no air bubbles at the bottom of the wells.
  7. Load the plate into a quantitative PCR thermal cycler and run using the cycles listed in Table 2.
    NOTE: Conventional qPCR protocols are typically limited to 40 cycles. Increasing the cycle count is unlikely to produce meaningful results, as by 40 cycles, a single target copy can theoretically yield approximately 1 trillion amplicons19.
StepTemperature (°C)Time (s)Cycles
Uracil-N-Glycosylase (UNG)  Inactivation501201
Denaturation951201
Denaturation95145
Annealing6020
Hold4∞1

Table 2: Thermocycler conditions for quantitative polymerase chain reaction. These conditions are attuned to a specific series of commercial gene expression assays. Default parameters are used, with the exception of increasing amplification cycles from 40 to 45.

7. Example volume calculation for qPCR

NOTE: A R source code for a volume calculator for the described equations below is available in Supplementary File 1. This calculator is for the Taqman probes and master mix listed in the Table of Materials.

  1. Determine the number of wells to be pipetted and calculate a minimum of 12.5% excess. This excess constant (kExcess) compensates for pipetting error and ensures there is enough solution. For this example, 96 wells will be used as the target for sample preparation.
    Equation:
    Equation showing well calculation process, formula: #Wells × k<sub>Excess</sub> = #Calculated wells.
    Example:
    Equation multiplying 96 wells by 1.125 excess to calculate 108 wells, relevant to lab experiments.
    NOTE: If 12.5% excess is not an integer, an easy way to ensure "nice" numbers in subsequent steps is to round up to the nearest well and adjust kExcess accordingly.
  2. Calculate the working master mix volume. In these experiments, 1 µL of cDNA and 9 µL of working master mix are used per well. cDNA and master mix volumes can be adjusted as needed.
    Equation:
    PCR master mix volume calculation formula; equation for lab use in preparing reactions.
    Example:
    96-well plate calculation formula for PCR master mix volume, indicating reagent preparation.
  3. Calculate the concentration modifier (kConc). This modifier is used to create a more concentrated working master mix that will dilute to 1x when mixed with cDNA.
    Equation:
    Equation for concentration ratio in microplate assay; final volume over master mix volume.
    Example:
    Micropipette dilution calculation, 10μL total/9μL mix per well, description of ratio formula.
  4. Calculate the probe (20x) volume.
    Equation:
    TaqMan probe volume calculation formula; equation for master mix dilution; PCR experiments.
    Example:
    Master mix calculation equation for TaqMan probe, PCR setup, molecular biology method.
  5. Calculate master mix stock (2x) volume.
    Equation:
    Calculating master mix stock volume; formula, dilution factor, biochemistry process diagram.
    Example:
    Master mix dilution calculation, formula: (972 μL/2) × (10/9) = 540 μL, molecular biology tutorial.
  6. Bring the working master mix up to the calculated volume using molecular-grade H2O.
    Equation:
    Working master mix volume (μL) equation; PCR preparation; gene amplification method.
    Probe 1 volume (µL) equation; research data analysis; lab experiment setup.
    Probe 2 volume measurement (µL) in chromatography experiment.
    Master mix stock volume (µL) formula, biochemical experiment setup.
    Equation showing water volume in microliters, denoted as H₂O volume (µL) in scientific notation.
    Example:
    Working master mix volume, 972 µL equation; used in PCR preparation, biochemical research.
    Probe volume equation, -54 µL of probe 1 (20x), relevant for laboratory calculations.
    Microscopic measurement, 54 µL probe 2 (20x) for sample analysis, chemical experiment setup.
    Master mix preparation formula, "-540 µL of master mix stock (2x)," used in PCR experiment.
    Static equilibrium, ΣFx=0 equation; scientific diagram; educational use; physics balance concept.

Supplementary File 1: A R source code for a volume calculator. Please click here to download this File.

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Results

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 ...

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Discussion

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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Centrifuge and RotorFisher Scientific14285554PM
Chloroform, HPLC gradeAlfa Aesar22920
Ethanol (190-proof)Decon Labs2801
Ethanol (200-proof)Fisher BioReagentsBP2818
Fisherbrand Pellet PestleFisher Scientific12-141-363
Forceps, Curved; Student Tweezers #7World Precision Instruments501981
Forceps, Straight; Dumont #5Fine Science Tools11252-40
Forceps, Straight; Student Tweezers #4World Precision Instruments501978
Kimwipes, smallKimberly-Clark34155Delicate task wipe
MicroAmp Adhesive Film ApplicatorApplied Biosystems4333183
MicroAmp EnduraPlate Optical 96-Well Fast Clear Reaction Plate with BarcodeApplied Biosystems4483485
MicropipettorsEppendorf 01-671-120
Microscope, DissectionCarl ZeissSteREO Discovery.V8
MiniAmp Optical Adhesive CoverApplied Biosystems4360954
MiniAmp Thermal CyclerApplied BiosystemsA37834
MixMate Plate Vortex MixerEppendorf5353000529
Molecular Grade WaterFisher ScientificBP28191
Nanodrop One Microvolume UV-Vis SpectrophotometerThermo ScientificND-ONE-W
Nunc MicroWell MiniTraysFisher Scientific12-565-154Dissection Tray
PCR TubesVWR201007-012
Petri Dish, 60 mm x 15 mmFisher ScientificFB0875713A
Phosphate Buffered Saline, 1x Dulbecco'sGibco14190-136
Pipette Tips, 10 µL GraduatedUSA Scientific1161-3700
Pipette Tips, 1250 µL XL GraduatedUSA Scientific1161-1720
Pipette Tips, 200 µL Profile GraduatedUSA Scientific1163-1700
QuantStudio 3 Real-Time PCR SystemApplied BiosystemsA28567
Reverse Transcriptase, SuperScript IV VILO Invitrogen11756050Highly processive and thermostable reverse transcriptase
RNA Clean and Concentrator 5 KitZymoZR1013
RNase-Free WaterFisher BioReagentsBP2819
RNaseZapSigmaR2020RNase decontamination solution
SealRite Microcentrifuge 1.5 mL TubesUSA Scientific1615-5500
SuperFine Vannas 8 cmWorld Precision Instruments501778Scissors
TaqMan FAST Advanced Master Mix for qPCRApplied Biosystems4444557
TaqMan Gene Expression Assay (FAM) Probe (MTO)ThermoFisher Scientific4448892
TRIzolInvitrogen15596026Acid-guanidinium-phenol solution
Vortex MixerThermo Scientific88880017

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Fiber Cell MassMouse LensLens Epithelial CellsLens Fiber CellsMicrodissectionSpin Column PurificationDifferential Gene ExpressionConnexin Expression
RNA Isolation from Mouse Ocular Lens Epithelium and Fiber Cell Bulk Masses
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