This protocol was designed to address a very important and unsolved issue on how microbial substances present and environmental allergens can regulate the development of allergic inflammation. Specifically, the methods presented here can help answer whether double-stranded RNA species in house dust mites are immunogenic in vivo and able to modulate eosinophilic lung inflammation. The main advantages of these techniques are that they're simple, efficient, highly reproducible and can be performed using commonly used tools and equipment.
These methods can also be used to evaluate lung diseases caused by respiratory viral infections. Li She, a graduate student in my laboratory, will help demonstrate the procedure. Begin by isolating total RNA from allergens, insects, and non-insect allergens.
Transfer a proper amount of specimen into a two-milliliter tube containing 1.4 millimeters of ceramic beads, and freeze the tubes in a liquid nitrogen container for approximately 10 minutes. Add one milliliter of guanidinium thiocyanate-based RNA isolation reagent to each tube. Then break the insect and non-insect small animals with a high-energy cell disrupter at maximum speed for 45 seconds.
Chill the sample on ice, and repeat this process twice. Transfer the solution into a new 1.5-milliliter tube. Add 200 microliters of chloroform to each tube, and vortex the sample.
Centrifuge the tubes at 14, 000 times g for 14 minutes at four degrees Celsius. Then transfer the upper aqueous phase into a new tube containing 500 microliters of isopropanol. Mix the sample.
Then centrifuge it at 14, 000 times g for 14 minutes at four degrees Celsius. Carefully aspirate the supernatant. Then wash the RNA pellet with 500 microliters of 75%ethanol, and centrifuge it at 7, 500 times g for 10 minutes at four degrees Celsius.
Carefully remove all liquid. Air-dry the pellet, and dissolve the RNA with 20 to 50 microliters of RNase-free water. To detect the double-stranded RNA structures in the total RNA, spot two microliters of the 200-nanogram-per-microliter RNA sample onto the positively charged nylon membrane.
Crosslink the samples to the membrane at 1, 200 microjoules by 100 in a UV crosslinker. Repeat the spotting and crosslinking two more times, which will result in a total of 1.2 micrograms of RNA per blot. Block non-specific binding with 5%milk in TBS-T for one hour while shaking at room temperature.
Then remove the blocking solution, and add the anti-double-stranded RNA J2 antibody at a one-to-1, 000 dilution in 1%milk in TBS-T. Incubate the membrane overnight with shaking at four degrees Celsius. On the next day, wash the membrane three times with TBS-T for five minutes per wash.
Add the secondary antibody, and incubate the membrane for one hour at room temperature. Then repeat the washes with TBS-T. Add the substrate, and incubate the membrane for five to 15 minutes until a desired signal is visible.
Stop the reaction by rinsing the membrane with double-distilled water. To collect the lung samples, place the lungs on tissue papers, and excise one small piece of each lung lobe. Place each piece into a two-milliliter tube containing beads.
To collect bronchoalveolar lavage, disinfect a euthanized mouse with 70%ethanol, and use scissors to cut the skin from the upper area of the abdomen to the neck. Gently pull the salivary glands and the sternohyoid muscle apart with forceps to expose the trachea. Then place a nylon string beneath it.
Make an incision in the trachea approximately two milliliters under the larynx just enough to insert a cannula, and knot the string around the trachea and cannula. Attach a syringe to the end of the cannula, and load it with fresh PBS-EDTA. Then inject and aspirate one milliliter of the solution into the lung.
Detach the syringe from the cannula, and eject the solution into a 15-milliliter tube. Repeat this process to ice with fresh PBS-EDTA, and pool the lavage. Centrifuge the tube at 500 times g for seven minutes at four degrees Celsius.
Then record the volume, and transfer the supernatant to two 1.5-milliliter tubes without disturbing the pellet. After resuspending the pellet, transfer 150 microliters into a 96-well plate, and centrifuge the plate for seven minutes at 500 times g and four degrees Celsius. Then quickly invert the plate on tissue paper to remove the supernatant.
Stain the cells with antibodies in FACS buffer in the presence of 2.4G2 blocking antibody, and incubate the plate at room temperature for 30 minutes in the dark. After staining, centrifuge the plate to pellet the cells at 500 times g for seven minutes at four degrees Celsius. Remove the staining solution by inverting the plate on tissue paper.
Add 100 microliters of FACS buffer to wash the cells, and then repeat the centrifugation. Resuspend the samples in 150 microliters of FACS buffer, and then transfer them to FACS tubes containing an additional 350 microliters of buffer. Add 25 microliters of counting beads to each sample, and proceed with flow cytometry analysis.
The presence of long double-stranded RNA structures in house dust mites, insects, and non-insect small animals was examined by dot blot using a double-stranded, RNA-specific mouse monoclonal antibody. RNase III was used to digest the double-stranded RNA into 12 to 15 base pair fragments, which were undetectable by J2.The ability of house dust mite total RNA to stimulate an innate immune response in mouse lungs was demonstrated by RT-qPCR. The RNase III treatment abolished the immunostimulatory activity of house dust mite total RNA, indicating that the double-stranded RNA structures are essential for innate immune activity in the lungs.
The inhibitory effect of house dust mite total RNA on the development of a severe type two lung inflammation were evaluated with FACS analysis. The eosinophilic lung inflammation was induced by dust mite extracts, which were treated with or without RNase III. The degradation of long double-stranded RNA species resulted in severe type two lung inflammation, reflected by the increased eosinophil numbers in bronchoalveolar lavage and lungs.
Notably, the number of eosinophils in the dust mite total RNA-treated group is comparable to the group treated with the original dust mite extract that endogenously contained the long double-stranded RNA species. When attempting this protocol, be very careful when injecting and recollecting the BAL fluid, as any damage to the lungs at this stage may alter the final results. In addition to this procedure, other methods like ELISA can be performed to analyze non-cellular soluble contents present in the BAL fluid.
After its development, this technique paved the way for researchers to explore other microbial substances present in environmental allergens, such as non-double-stranded RNA factors that can regulate the development of allergic lung inflammation.
