The overall goal of this procedure is to analyze the expression of micro RNA in microglia. This is accomplished by first dissecting the brain and spinal cord and then homogenizing the central nervous system or CNS tissue. In the second step, mononuclear cells from the CNS are isolated by a per call gradient, and then the microglial population is identified by facts.
Next, the microglial RNA is isolated and the quality of the sample is analyzed. Finally, real-time PCR for the particular micro RNA of interest is performed. Ultimately, the delta delta CT method can be used to show the relative level of expression of a particular micro RNA in microglia.
Generally, individuals new to this method will struggle because they fail to isolate sufficient number of mononuclear cells from the CNS or because the sample is contaminated, myelin and cell debris. Both cases result in low quality preparation, but either low concentration of RNA or contamination with protein respectively. Demonstrating the procedure will be me and vir.
Dr.O fellow from After perfusion of all the mice, dissect the brains and spinal cords. Then place one brain and one spinal cord at a time into a 15 milliliter downs homogenizer adding five milliliters of PBS to the tissues each time. After performing 10 to 20 gentle strikes, pass the homogenous through 70 micron cell strainers into individual 50 milliliter conical tubes and centrifuge the tubes for five to seven minutes at 834 times G and four degrees Celsius.
After discarding the supernatant Resus suspend the brain and spinal cord homogenous from two to three mice in five to seven milliliters of 70%per call. Then overlay seven milliliters of 40%per call and spin the per call and cell solution for 30 minutes at 850 times G 20 degrees Celsius with no break. Discard the damaged cells and mile and debris at the top of the 70%per call.
Then collect the mononuclear cells at the interface between the 70%and 40%per call solutions and dilute the collected cells in PBS, at least at a one-to-one ratio. After spinning down the cells, resus the pellet in half a milliliter of PBS and transfer them into a 1.7 milliliter einor tube. Begin by transferring 50 microliters of the density gradient sorted cells into a new 1.7 milliliter einor tube and add 350 microliters of fax buffer to the tube.
Then after adding five to 10 microliters of anti FCR antibodies to the tube, incubate the cells for 10 to 15 minutes on ice. Split the cell sample into two einor tubes to one tube. Add one microliter of anti CD 11 B and two microliters of anti CD 45 antibodies.
Incubating the cells for 10 to 15 more minutes on ice. Label the second tube negative control and place it on ice. After incubation with staining antibodies, add one milliliter of fax buffer to both tubes and spin down the cells in a micro centrifuge for five minutes at 1200 times.
G.Then fix the pellets in 400 microliters of 1%para formaldehyde in PBS and vortex the tubes to prevent clumping. Finally, analyze the cells by flow cytometry using the negative control cell sample and BD com beads begin by subcutaneously immunizing a group of five to 10, eight to 12 week old C 57 black six mice with 150 milligrams of MOG 35 to 55 peptide in four milligrams per milliliter of complete frons adjuvant to induce EAE. Then administer pertussis toxin intraperitoneal on day zero and on day two post immunization.
After isolating and staining the cells as just shown resus, suspend the pellets in 400 microliters of PBS instead of fixative just prior to sorting. Then fill two 1.7 milliliter einor tubes with 300 microliters of PBS supplemented with 10%FBS for collecting the samples. Finally, sort the microglia and macrophage populations to a high purity using the gating strategy illustrated in this representative density plot.
After sorting, reanalyze the microglia and macrophage populations to determine the purities of the isolated cell samples. After spinning down the sorted cell populations at 1200 times G for five to seven minutes in a micro centrifuge, carefully remove the supernatant and freeze the cell pellets on dry ice. Next, transfer the frozen cell pellets to regular ice.
For thawing, add 300 microliters of lysis buffer from a Nirvana kit and then gently mix the cell solution by pipetting five to seven times. Then add 30 microliters of the homogenate additive, vortex the cell suspension for 30 seconds, and then place the tubes on ice for 10 minutes. Now add 300 microliters of the acid phenol, chloroform, and vortex the tube for 30 more seconds.
Then after spinning down the cells for five minutes at 9, 500 times G, carefully transfer the upper 300 microliters of the supernatant containing the aqueous phase to a new 1.7 milliliter einor tube. Mix the solution with 375 microliters of 100%ethanol. Transfer the entire tube contents to a filter cartridge from the Nirvana kit and spin the cartridge for one minute at 9, 500 times G.Next, discard the flow through buffer.
Add 700 microliters of wash solution one from the Nirvana kit, and spin for another minute at 9, 500 times G after washing the cartridge. Two more times with 500 microliters of wash solution. Two from the Nirvana kit Elute, the RNA from the cartridge with 60 microliters of preheated nuclease free water.
Finally, use a NanoDrop spectrophotometer to assess the purity and quantity of the RNA after performing realtime Q-R-T-P-C-R to detect micro RNA in the microglia and macrophage samples. P-R-T-P-C-R curves for fluorescently labeled mere 1 24 and snow RNA 55 PCR products can be created as shown here in the first graph. Typical curves for the amount of fluorescently labeled specific PCR products for control snow RNA 55 RNA samples isolated from microglia or bone marrow derived macrophages as compared to the number of PCR cycles are shown.
Note how the CT values for analysis of the snow RNA 55 expression from the different cell populations are determined at the intersection of the baseline with the lower part of the linear range of the Q-R-T-P-C-R curves as indicated in the graph by the green and blue vertical lines. In this second graph, the amount of fluorescently labeled specific PCR products for the experimental mere 1 24 RNA samples from microglia or bone marrow derived macrophages compared to the number of PCR cycles is shown. Note how the CT values for analysis of the mere 1 24 expression from the different cell populations are again determined at the intersection of the baseline with the lower part of the linear range of the Q-R-T-P-C-R curves as indicated in the graph by the green and blue vertical lines.
After determining the CT values for each of the samples, as for the snow RNA 55 and mere 1 24 expression in microglia and BM dms from normal and EAE mice shown in this table, subtract the CT value for the control snow RNA 55, sample from the CT value for the experimental mere 1 24 sample to calculate the delta CT values for each duplicate of each sample. Then subtract the delta CT of the reference sample from the delta CT values of the other samples to calculate the delta delta CT values. Finally, the relative level of expression for each sample is calculated as two to the power of minus delta delta CT as seen in this density plot.
In the case of a good isolation of microglia from normal CNS, 95 to 98%of the cells will represent CD 11 B positive CD 45 low microglia with two to 5%of the cell suspension being made up of CD 11 B positive CD 45 high perivascular macrophages less than 1%of CD 11 B negative CD 45 high lymphocytes or CD 11 B negative CD 45 negative astroglia or oligo DRO glial cells or cell fragments will be present comparatively in a bad preparation. There is a significant contamination of CD 11 B, negative CD 45 negative cells or cell fragments such as astroglia or myelin particles making the cells unsuitable for RNA isolation and further analysis. In addition, CD 11 B negative CD 45 high cells also may be present indicating contamination by blood lymphocytes due to insufficient perfusion In this density plot, the three populations of the cells present in the CNS during neuroinflammation CD 11 B positive CD 45, low microglia CD 11 B positive CD 45 high macrophages and CD 11 B negative CD 45 high lymphocytes are shown in this figure.
The quality of RNA as determined by gel electrophoresis after a good versus bad isolation is shown. When the RNA is isolated in a clean manner, an RNA ladder and ribosomal RNA are evident when the isolation process is performed poorly smear and low molecular weight degradation products are evident. These final data demonstrate expression of mere 1 24 in normal adult microglia microglia of mice with EAE and microglia isolated from mice during early stages of development.
In this first bar graph, the relative expression of mere 1 24 is much greater in microglia from normal CNS compared to that observed for bone marrow derived macrophages. Similarly, although there is an increase in mere 1 24 RNA in macrophages from the CNS of mice with EAE microglia still express five times the amount of mere 1 24 RNA. This final bar graph demonstrates how changes in the level of expression of mere 1 24 in microglia during the development of mice of one, two, and four weeks of age as compared to adult eight week old mice also can be evaluated using this method While attempting the procedure.
It is important to remember to have a sufficient quantity and good quality of isolated cells, which will guarantee good results for the entire experiment.
