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

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.