After collecting mouse tears using Schirmer strip fragments, place the strips in a 600 microliter micro centrifuge tube containing 20 microliters of sterile water. Puncture the tip of the tube containing the sample, and transfer it to a new sterilized 1.5 milliliter tube. Centrifuge the tube at 2, 000G for 20 minutes at 4 degrees Celsius.
Then, pull the samples to get a total volume of approximately 22 microliters of supernatant per mouse. Place the tubes on dry ice to prevent biomolecule degradation. Next, add 200 microliters of a monophasic solution of phenol and guanidinium thiocyanate homogenized by pipetting, and allow the mixture to stand for five minutes at room temperature.
Then, add 40 microliters of chloroform. Vortex for 15 seconds and incubate the mixture for two minutes at room temperature. After that, centrifuge at 10, 000G for 15 minutes at 4 degrees Celsius.
Carefully transfer the aqueous phase to a new 1.5 milliliter tube without disturbing the interphase. Next, add 0.5 microliters of glycogen to 100 microliters of isopropanol and mix by pipetting. Allow the mixture to stand for 10 minutes at minus 20 degrees Celsius.
Then, centrifuge at 10, 000G for 10 minutes at 4 degrees Celsius. Quickly decant the supernatant. Add 200 microliters of cold 75%ethanol and vortex the tube to resuspend the RNA pellet.
Centrifuge at 8, 000G for five minutes at 4 degrees Celsius. After removing ethanol and drying the tube for 20 to 30 minutes, add 20 microliters of RNase-free water and resuspend the pellet. Then, measure the optical density in the micro volume spectrophotometer at 260 and 280 nanometers to assess the purity of RNA.
To synthesize cDNA from Tear RNA, add 10 to 5, 000 nanograms of RNA. Mix it with one microliter of oligo dT and up to 12 microliters of RNase-free water. Spin at 500G for 30 seconds and incubate at 65 degrees Celsius for five minutes.
Then, add four microliters of reaction buffer, one microliter of RNase inhibitor, two microliters of dNTP mix, and one microliter of reverse transcriptase to the mixture. After spinning the tube, incubate at 42 degrees Celsius for 60 minutes. After that, incubate at 70 degrees Celsius for 10 minutes to conclude the reaction.
Use the program and primers shown here for the qPCR cycle. Perform a milk curve analysis to check for the presence of primer dimers or unspecific amplicons in the reaction. The qPCR revealed CT values between 20 and 24, a suitable range for the starting amount of target mRNA in the sample.
However, the melt curve analysis suggested the low presence of target mRNA. The 2%agarose gel confirmed the presence of amplicons at the expected size.
