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

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 manner^1,2,3. 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 objects^{5,6,7,8,9,10,11} and maintain tissue transparency, and the innermost fiber cells are eventually compacted, resulting in a rigid lens center^9,12. 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.

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.

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 unclear^16,17. 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).

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.

Figure 2: A step-by-step RNA isolation workflow diagram.

(A) Phase separation: steps 3.1–3.11. These steps depict the process of homogenizing primary tissue, extracting RNA, and isolating RNA via an acid-guanidinium-phenol phase separation. (B) RNA concentration: steps 3.12–3.19. These steps depict the washing and concentration of isolated RNA using clean-and-concentrator columns. (C) Elution: steps 3.20–3.26. These steps depict eluting the RNA from the clean- and-concentrator columns using RNase-free water and heating to promote solubilization. Please click here to view a larger version of this figure.

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 × 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 × 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 × 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 × 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 × g for 1 min at 4 °C.

19. Discard the flowthrough and centrifuge once more at 16,000 × 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 × 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.

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.

Step	Temperature (°C)	Time (min)	Cycles
Annealing	25	10	1
Reverse Transcription	50	10	1
Enzyme Inactivation	85	5	1
Hold	4	∞	1

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.

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.

Step	Temperature (°C)	Time (s)	Cycles
Uracil-N-Glycosylase (UNG) Inactivation	50	120	1
Denaturation	95	120	1
Denaturation	95	1	45
Annealing	60	20
Hold	4	∞	1

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 $\left(k_{Excess}\right)$ 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:

   $$\text{\#}Wells\times k_{Excess}=\text{\#}Calculatedwells$$
   Example:

   $$96wells\times 1.125excess=108calculatedwells$$

   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 $k_{Excess}$ 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:

   $$\begin{array}{l}\text{\#}Wells\times k_{Excess}\times Workingmastermixvol.perwell(\mu L)\\ =Workingmastermixvolume(\mu L)\end{array}$$
   Example:

   $$96wells\times 1.125Excess\times 9\mu L=972\mu Lofworkingmastermix$$

3. Calculate the concentration modifier $\left(k_{Conc}\right)$. This modifier is used to create a more concentrated working master mix that will dilute to 1x when mixed with cDNA.

   Equation:

   $$\frac{Finalwellvolume(\mu L)}{Workingmastermixvolumeperwell(\mu L)}=k_{Conc}$$
   Example:

   $$\frac{10\mu Ltotalperwell}{9\mu Lofworkingmastermixperwell}=\frac{10}{9}=1.\overline{11}$$

4. Calculate the probe (20x) volume.

   Equation:

   $$\frac{Workingmastermixvolume(\mu L)}{Dilutionfactor}\times k_{Conc}=TaqManprobevol\text{.}(\mu L)$$
   Example:

   $$\frac{972\mu Lofmastermix}{20}\times \frac{10}{9}=54\mu LofeachTaqManprobe$$

5. Calculate master mix stock (2x) volume.

   Equation:

   $$\frac{Workingmastermixvolume(\mu L)}{Dilutionfactor}\times k_{Conc}=Mastermixstockvolume(\mu L)$$
   Example:

   $$\frac{972\mu Lofmastermi\text{x}}{2}\times \frac{10}{9}=540\mu Lofmastermixstock$$

6. Bring the working master mix up to the calculated volume using molecular-grade H₂O.

   Equation:

   $$\begin{array}{l}Workingmastermixvolume(\mu L)\\ \text{−}probe1volume(\mu L)\\ \text{−}probe2volume(\mu L)\\ \text{−}Mastermixstockvolume(\mu L)\\ =H_{2}Ovolume(\mu \text{L})\end{array}$$
   Example:

   $$\begin{array}{l}972\mu Lofworkingmastermix\\ -54\mu Lofprobe1(20x)\\ -54\mu Lofprobe2(20x)\\ -540\mu Lofmastermixstock\text{(}2x\text{)}\\ =324\mu L{H}_{2}O\end{array}$$

Representative 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 650.0 ng/μL (SD ± 152.2) for the epithelium and fiber samples, respectively. Using 5 ng/well reactions, this theoretically allows for an average of 160 reactions from a single pair of lens epithelium isolated using this protocol. The average RNA purities measured by A260/280 ratios were 1.92 (SD ± 0.05) for epithelium and 2.05 (SD ± 0.01) for fibers, indicating minimal protein contamination. Generally, an A260/280 ratio of ~2.0 is considered pure for RNA²⁰. Overall, this isolation yielded a sufficient concentration of highly pure RNA to reverse transcribe to cDNA and perform repeated qPCR experiments.

We performed reverse transcription and qPCR as described in the protocol. We chose 7 genes with known high expression of transcripts or proteins in the lens for real-time qPCR analysis to reveal differential expression between tissue compartments^{21,22,23,24,25,26,27,28,29,30,31,32}. Relative expression is shown as 2^−ΔΔCq values, normalized to epithelial cells (Figure 3). Mean relative expression is shown as geometric means with standard deviations. Due to the known differential expression, connexins were used to determine the successful separation of epithelium and fiber cells. Connexin 43 (Cx43; gap junction protein alpha 1; Gja1) is expressed in lens epithelial cells³³, Cx46 (Gja3) is expressed in lens fiber cells³⁴, and Cx50 (Gja8) is expressed in both epithelial and fiber cells^25,29. Lens fibers are observed to express Gja1 and Gja8 at approximately 200- and 7.5-fold lower levels than the lens epithelium, respectively. Meanwhile, Gja3 is approximately 1.5-fold more highly expressed in the lens fibers, consistent with previous reports²⁸.

Figure 3: Relative gene expression comparing isolated lens epithelium to fiber cell bulk mass.

RNA levels are normalized to the epithelial compartment. Epithelial cell markers Gja1 [encode for connexin 43 (Cx43)], Chd1 (encodes for E-cadherin), and Pax6 (encodes for transcription factor Pax6) show elevated expression in epithelial cells compared to fiber cells. Fiber cell markers Gja3 (encodes for Cx46) and Crygs (encodes γS-crystallin) show elevated expression compared to the epithelium. Markers expressed in both epithelium and fiber cells, Gja8 (encodes for Cx50) and Chd2 (encodes for N-cadherin), show detectable signal in both compartments. Plots show geometric means with standard deviations using the 2^−ΔΔCq method. Statistical significance was determined using two-way ANOVA followed by the Šidák multiple comparison test. * p ≤ 0.05; ** p≤ 0.01; *** p≤ 0.001; **** p≤ 0.0001. Please click here to view a larger version of this figure.

Similarly, differential expression was observed with cadherins, namely E-cadherin (Cdh1) and N-cadherin (Cdh2). E-cadherin protein expression is restricted to the lens epithelium while N-cadherin is expressed in both the epithelium and fibers³⁵. Here we show expression of Cdh1 and Cdh2 are approximately 330-fold and 2.4-fold lower in fibers compared to the epithelium.

Two additional epithelial and fiber cell markers, paired box 6 (Pax6) and γS-crystallin (Crygs), respectively, were also used to demonstrate successful separation of the lens compartments^36,37. Consistent with previously observed expression patterns, Pax6 is restricted to the epithelial cells, exhibiting 8-fold lower expression in the fibers. Gamma-crystallin proteins are expressed primarily in lens fiber cells, and we observe Crygs is approximately 3-fold higher in the fibers compared to the epithelium¹. These data indicate a clean separation of lens epithelium and fiber mass, allowing transcriptomics analysis of each tissue compartment for comparison.

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 fibrous bulk mass compartment to avoid contaminating epithelial cell samples with significant amounts of fiber cells. Lens epithelial cells are tightly adhered to the capsule, and the adhesion between epithelial cells and underlying fiber cells is relatively weak, allowing clean isolation of the capsule and attached epithelial cells from the fiber cell mass. Once the lens compartments have been isolated and deposited into TRIzol, samples should be processed in a chemical fume hood to avoid noxious fumes and to prevent contamination and degradation of the isolated RNA.

The RNA isolation process uses a modified commercial protocol from a RNA Clean and Concentrator 5 Kit. Previously, isolation of sufficient concentrations of high-purity RNA from epithelial cells was difficult due to the small number of starting cells from each lens. The use of the RNA concentrator kit has made it possible to isolate RNA from pairs of lens epithelial sheets. From our testing, we excluded the RNA-binding and DNase digestion steps from the commercial protocol and added a short incubation step prior to RNA elution from the spin column to increase RNA yield from lens epithelial cell samples. This has been observed to increase yield while still producing clean RNA samples with high purity. The omission of the DNase was accommodated by selectively using exon-spanning TaqMan probes when available to avoid amplifying genomic DNA. Notably, the RNA integrity number (RIN) of our isolated epithelial and fibrous RNA fell between 6.4–7.4 and 4.8–5.2, respectively, indicating that this approach may be suitable for techniques such as RNA sequencing.

As we tested this protocol, we could isolate RNA from single lens capsules with attached epithelium. On average, the RNA yield from one lens capsule from an adult mouse sample is about half of the yield from two capsules. This would make it possible to use single lenses for sample collection from experiments when one eye may be treated and the contralateral eye is the untreated control. It may also be possible to increase RNA concentration from single capsule isolations by decreasing the volume of the elution buffer. While the concentration of RNA from epithelial cell samples is sufficient for a sensitive method like qPCR, for other types of RNA analysis, e.g., RNA sequencing, pooling of more than 2 lens capsules may be required to isolate enough starting materials. Pooling of specimens may also be required for research models with smaller lenses, such as perinatal mice or zebrafish, to generate enough material for downstream analysis. Working with perinatal mice may also prove challenging, as, depending on age, the hyaloid arteries that temporarily supply the developing lens with blood may not have regressed, requiring more thorough cleaning to prevent contamination of extralenticular tissue. For non-mouse lenses, it should be noted that while murine lens capsules successfully dissolve and homogenize in TRIzol, additional mechanical perturbation may be required for larger specimens such as human lens capsules.

These methods allow for a higher resolution, more nuanced perspective of the lens in molecular and biochemical studies. The ability to separate the epithelium from the lens fibers provides the opportunity to better understand the changes between epithelial cells and fiber cells as they mature. A similar method was presented in Parreno et al. (2022), where RNA isolated from epithelium was compared to immortalized cell lines²⁷. The presented method differs slightly in RNA isolation and concentration stages, omitting RNA binding buffer and DNase digestion steps to achieve a higher yield while maintaining purity. We also used the RNA concentration kit on lens fiber cell masses to improve RNA purity for subsequent qPCR experiments. This improved method was implemented to investigate expression differences of Ephs and ephrins in the ocular lens of EphA2 and ephrin-A5 mutant mice at different age points³⁹. Another approach presented by Zelenka et al. (2009) details a similar separation of lens epithelium from fibers in neonatal rats, followed by explant culture and differentiation by stimulation with fibroblast growth factor 2 (FGF-2)⁴⁰. The methods described here present a simplified workflow that allows investigation of native epithelial cells rather than cultured and differentiated explants.

Due to the lifelong growth of the lens and lack of cell turnover, the presented method allows for spatial and temporal snapshots of gene expression by investigating the different compartments of the lens. This allows for a more detailed look at the biological processes involved in lens maintenance and maturation, and the comparison of proliferative epithelial cells to maturing fiber cells. The successful implementation of the presented method and similar methods indicate that this protocol can be modified and optimized for mice of varying ages and genotypes. Moreover, the ability to isolate sufficient material for transcriptomic investigations from mouse lenses indicates that this protocol should be applicable to other animal models with similarly sized or larger lenses or to human lens capsule samples collected during cataract surgery. The ability to isolate the different cell types of the lens enables a deeper understanding of the diverse roles these cells play in the lens.

Supplementary Material

R source code for a volume calculator

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