This method can help answer key questions in therapy of microRNA to the kidney, which remains a challenge. The main advantage of this technique is that it's enabled the therapy of microRNA mimics to the kidney without causing an interferon response or genetic instability. Begin by dissolving 10 microliters of PEI-NPs in 90 microliters of 5%glucose solution in a 1.5-milliliter microcentrifuge tube.
Then vortex the tube gently and spin down. Mix 50 microliters of artificially synthesized miRNAs dissolved in 100 micromolar of nuclease-free water with 50 micromolar of 10%glucose solution in 1.5-milliliter microcentrifuge tubes. Vortex the tube gently and spin down.
This process yields miRNA dissolved in 5%glucose solution. Then mix 100 microliters of PEI-NPs in 5%glucose solution and 100 microliters of miRNA mimic in 5%glucose solution. Vortex the tube gently and spin down.
Incubate the mixture for 15 minutes at room temperature to prepare a stable complex of PEI-NPs miRNA mimic. Dissolve two microliters of PEI-NPs in 98 microliters of 10%glucose solution in a 1.5-milliliter microcentrifuge tube. Vortex the tube gently and spin down.
Then add 100 microliters of Cy3-labeled miRNA mimic to 100 microliters of PEI-NPs in 10%glucose solution and vortex the tube gently. Incubate the mixture for 15 minutes at room temperature to prepare a stable complex of PEI-NPs-Cy3-labeled-miRNA-mimic. Place the UUO mouse headfirst in a 50-milliliter centrifuge tube without anesthesia and then place the tail through a prepared hole in the cap.
Fill a one-milliliter syringe with a 30-gauge needle with 200 microliters of PEI-NPs-Cy3-labeled-miRNA-mimic complex and inject the complex via the tail vein of the mouse. Next, make an incision into the skin, muscles, and ribs with surgical scissors and forceps to expose the heart. After making an incision into the right atrium, inject PBS into the left ventricle until the kidney color changes to pale yellow.
This indicates that the whole mouse body is perfused with PBS. After collecting the kidneys, remove the renal capsules, and wash them twice with PBS. Embed the kidney tissue samples in OCT compound and freeze them in liquid nitrogen.
Use a cryostat to prepare sections and mount the sections onto silane-coated glass slides. Fix them with 4%paraformaldehyde and gently wash the slides twice with PBS. Finally, visualize the fluorescence staining by fluorescence microscopy at 100x and 400x magnification and appropriate imaging software.
The representative image shows the structure of the PEI-NPs-miRNA-mimic complex. The delivery and effects of miRNA-146a-5p-mimic using PEI-NPs in renal fibrosis are depicted in these images. Fluorescent microscopy analysis showed that tail-vein injection of PEI-NPs could deliver the miRNA mimic to the tubulointerstitial space.
In renal fibrosis mice produced by UUO, this included renal tubular cells which do not have a regular form in the kidney and the glomerulus. qRT-PCR analysis showed that injection of PEI-NPs-miRNA-146a-5p-mimic induced significant overexpression of miRNA-146a-5p in the kidney. The histological analysis with Sirius red staining is shown here.
Fluorescent microscopy analysis showed that tail-vein injection of PEI-NPs could deliver miRNA mimic to the glomerulus and tubulointerstitial space in kidneys of diabetic kidney model mice in vivo. In addition, the qRT-PCR analysis showed that injection of PEI-NPs-miRNA-181b-5p-mimic induced significant overexpression of miRNA-181b-5p in the kidney. Histological analysis with Periodic acid-Schiff staining showed that injection of PEI-NPs-miRNA-181b-5p-mimic once per week from 10 to 20 weeks of age inhibited the progression of diabetic kidney disease in mice.
Delivery and effects of PEI-NPs-miRNA-5100-mimic in acute kidney injury model mice produced by ischemia reperfusion injury are shown here. The data of the fluorescent microscopy analysis qRT-PCR analysis to evaluate the overexpression effects of miRNA-5100 following injection of PEI-NPs-miRNA-5100-mimic and the histological analysis of hematoxylin-eosin stained kidney are depicted in these images. PEI-NPs injected via the tail vein two days before the ischemia reperfusion injury, or IRI, procedure prevent acute kidney injury progression induced by IRI.
When performing this technique, prepare five-micrometer thick sections which include as much kidney tissue as possible. You can also prepare seven-micrometer thick sections.
