The overall goal of this procedure is to locally apply IRNA via intrathecal delivery into the spinal cord. This is accomplished by first preparing the optimal I-R-N-A-P-E-I complex reagent. The second step is to prepare the mouse for intrathecal injection.
After at least 15 minutes of incubation time, the SI RNA mixed solution is drawn into a Hamilton syringe. The final step is to carefully perform the intrathecal injection, repeating the injections once every 24 hours to ensure optimal downregulation. Ultimately immuno blotting and quantitative real-time.
PCR are used to show the successful downregulation in protein and mRNA levels from the INA transfection. The main advantage of this technique is that it is less invasive than an intra catheter or a spinal cord injection. Generally, individuals new to this method must work to acquire the finest needed to inject through a small groove between the federal column without damaging the spinal cord.
To prepare the SIR a solution begin by diluting the desired amount of SIR A with sterile water. Then dilute this further with 10%glucose solution mixed by pipetting up and down. To make the PEI solution dilute the required amount of PEI reagent with sterile water and dilute this further with 10%glucose solution mixed by pipetting up and down.
Next, add the Irna solution to the PEI solution all at once and vortex gently then incubate the mixed solution for 15 minutes at room temperature while the mixture is incubating, prepare the mouse for intrathecal injection. After anesthetizing the mouse with 3%ice of fluorine, shave around two square centimeters of fur near the base of the tail and clean the injection site with alcohol. Then place the mouse in a nose cone for continued isof fluorine administration during the procedure and reduce the isof fluorine to 1.5%Using a 25 microliter Hamilton syringe attached to a 30 gauge half inch needle, draw up enough of the irna PEI solution to inject between five and 10 microliters.
Next, locate the most prominent spinous process, which is the L six. Fix the vertebral column around this area by gently pressing just above the L six, applying enough pressure to feel the spinous process without depressing the entire body. Then using a fingernail, locate the groove of the L five and L six vertebrae.
Now carefully insert the needle between the groove of the L five and L six vertebrae and look for a tail flick, which indicates a successful entry of the needle into the intradural space. Once the tail flick is observed, immediately but carefully secure the needle position with one hand and slowly inject between five and 10 microliters of substance with the other hand. Once the injection is finished, move the mouse back to the cage to recover from anesthesia.
Repeat this injection at least two more times every 24 hours to achieve optimal downregulation of the targeted gene. Following acute intrathecal injection with fast green dye, there is no visible sign of tissue injury at the site of the injection, which is marked by the die Punta excision of the spinal cord. A few minutes after injection shows a diffusion of dye in the rostral direction.
The star marks the injection site and the white box marks the lumbar region. A transverse view of the spinal cord at the L three to L five segment shows specific staining on the surface, but not the interior of the spinal cord. Following intrathecal injection of fast green intrathecal injections on three consecutive days of irna targeted against wave one mixed with PEI reagent resulted in a downregulation of the wave one protein in the dorsal lumbar spinal cord as compared to scramble siRNA control injections as shown by Western blotting and by QR TPCR.
Western blotting quantification from the lysate of ventral L three to L five spinal cord and cervical spinal cord showed similar downregulation of the wave one protein while lysates from the brain and DRGs did not. After watching this video, you should have a good understanding of how to do an intra injection efficiently and moreover to specifically downregulate proteins in the spinal cord.
