The overall goal of this RNA Transfection assay is to use HSP27 specific small interfering RNA to assess the potential role of HSP27 in corneal epithelial wound healing. This method can help answer key questions regarding the the function of Heat Shock Protein 27 during epithelial wound healing process. The main advantage of this technique is that small interfering RNA is sensitive RNA sucrose created to specifically target a messenger RNA transcript to induce it's degradation.
It has been used to identify novel pest space various cellular processes. Though this technique can provide the insight into the corneal epithelial wound healing, it may also apply to another system such as skin and mucosa. Demonstrating the procedure will be Soon-Suk Kang, a technician from my laboratory.
To begin this procedure, culture human corneal epithelial cells in a six-well plate with with bronchial epithelium growth medium at 37 degree Celsius and five percent carbon dioxide until they 95 percent confluence. Next, dilute 2.5 micro liters, or 7.5 micro liters of the transfection re-agent with 100 micro liters of reduced serum medium for transfection. Then, dissolve HSP27 specific siRNA and scrambled controlled siRNA in 100 micro liters or reduced serum medium to create 10 nanomolar of HSP27 specific siRNA and 15 nanomolar of scrambled control siRNA respectively.
Following that, mix 100 micro liters of siRNA solution with 100 micro liters of diluted transfection reagent and incubate the mixture for 15 minutes at room temperature. Subsequently, add siRNA lipid complexes to the cells. After a four hour incubation, change the medium to the complete bronchial epithelium growth medium, and incubate the cells two days at 37 degrees Celsius before western blot analysis.
In this step, extract HSP27 specific and scrambled control siRNA transfected HCECs in a biological safety cabinet using 100 micro liters of ice cold lysis buffer. Then incubate the cells for 30 minutes on ice to induce cell lysis. Next, centrifuge the lysates at 10, 000 times G for 15 minutes.
Subsequently, transfer the supernatants to fresh 1.5 milliliter tubes and store them at minus 80 degrees Celsius. After that, determine the protein concentrations of the cell lytates using the Bradford protein assay. Then load the samples with equal amounts of total cell proteins on a 10 percent or 12 percent acrylamide gel.
Subject the gel to SDS page, and electrophoretically transfer the separated protein bands to the nitrocellulose filters with 200 milliamps of current for one hour and four degrees Celsius. Next, block the nitrocellulose filter membranes with five percent skimmed milk in TBST for one hour. Then add primary anti-bodies and incubate the membranes overnight at four degrees Celsius on a shaker.
The next day, wash the membranes three times with TBST allowing ten minutes for each wash. Then detect the immunolreactive bands using horseradish peroxidase conjugated goat anti-rabbit antibodies in five percent BSA. Subsequently, incubate the membranes in the western blotting luminol reagents for one minute at room temperature.
Remove the membrane from the reagent solution. Then, remove excess liquid with an absorbent towel and place them in a plastic sheet protector. Afterward, place the covered membrane in a film cassette with the protein side facing up.
Place an x-ray film on top of the membranes and expose it for one minute. In a biological safety cabinet, make a wound by dragging a sterile pipette tip across the confluent cultures of HSP27 specific or scrambled control siRNA transfected HCECs. Immediately after wounding wash the cells twice with 1XPBS and maintain them in bronchial epithelium growth medium at 37 degrees Celsius and five percent carbon dioxide for 24 hours.
24 hours after wounding take the HCECs image using an upright microscope at 100x and perform background flattening using the filter command in image analysis software. Using the select measurements command, define the area of interest with the same sized polygonal shape that can cover the initial wound perpendicularly from end to end. Then, determine three different area of interest in the wounded area of each sample.
Automatically count the cell number in each field using the count size measure menu option. Western blot analysis shown here, revealed that the expression of phosphorylated HSP27 and phosphorylated Akt were both significantly reduced. Where as the expression of backs was significantly increased in HSP27 specific siRNA transfected HCECs.
The phosphorylated HSP27 expression was reduced by 30 percent and 40 percent in 10 nanomolar and 15 nanomolar of HSP27-specific siRNA transfected cells respectively, compared with control siRNA transfected cells. Moreover, the nonphosphorylated HSP27 expression was reduced by 20 percent and 30 percent in ten nanomolar and 15 nanomolar of HSP27 specific siRNA transfected cells respectively. The scratch induced directional wound assay indicated that 24 hours after wounding, HSP27-specific siRNA transfected cells at 10 and 15 nanomolar exibited reduced migration.
In addition HSP27-specific siRNA transfected HCEC's underwent more apoptotic and necrotic cell death compared with the scrambled control siRNA transfected cells shown by flow cytometry. Once mastered RNA transfection technique can be done in 24 hours if it is performed properly. Following these procedures an episode like lock mouth can be generated in order to answer additional question like the initial function of Heat Shock Protein 27 in cornea epithelial wound healing.
After watching this video, you should have a good understanding of how to use Heat Shock Protein 27 specific small interfering RNA to assess the potential role of Heat Shock Protein 27 in cornea epithelial wound healing. Don't forget that working with small interfering RNA and reagents can be hazardous and precursors such as a biological safety cabinet and protective mask should always be used while performing these procedures.
