RNA-Isolierung aus zellspezifische Subpopulationen Mit Laser-Mikrodissektion In Verbindung mit Rapid-Immunmarkierung

The overall goal of this procedure is to demonstrate how to perform rapid immuno labeling of a mouse brain cell population isolated by laser capture micro dissection or LCM while maintaining the RNA integrity of the samples. This is accomplished by first rapidly freezing the dissected brain tissue. The brain is then cut into thin sections onto RNA free glass microscope slides and treated with a quick immuno staining.

Finally, the cell population of interest is isolated using LCM. Ultimately, the quick staining followed by laser capture microdissection allows the identification of changes in the expression of the gene of interest in the experimental cell sample. The main advantage of this technique over existing methods like fluorescent activated cell sorting, is that with our technique, the spatial resolution is maintained allowing solution of the specific cell of ES from the surrounding tissue.

Generally, people new to this smith cell will circle because this procedure includes several critical steps that require working quickly to maintain the RNA integrity in the samples. To prepare the samples for sectioning, dissect the brains from day one postnatal mice in ice cold L 15 medium within two minutes of sacrifice. Next place the brains into embedding molds in the appropriate orientation for sectioning and fill the molds with frozen tissue, embedding media.

Then immediately freeze the specimens in liquid nitrogen and store them at minus 80 degrees Celsius within 30 seconds of snap freezing. Now, treat membrane coated glass slides with surface RNA decontamination to remove any trace amounts of RNA. Rinse them in DEPC water and let them air dry when the slides are dry.

Set the cryostat chamber temperature to minus 20 degrees Celsius and transfer the frozen sample to the cryostat. For each sample, mount the frozen block onto the specimen clamp and slice the specimens at a 10 micron thickness, collecting the sections onto the membrane coated slides. After letting the sections dry for at least 10 minutes, use a hydrophobic barrier pen to encircle the tissues.

After about 10 seconds, place the slides in freshly prepared cold fixative solution for no more than five minutes at minus 20 degrees Celsius. Now transfer the slides to a clean tray pretreated with surface RNA decontamination solution. First, shake the slides once to remove any excess fixative and wash them with six to eight quick dips In D-E-P-C-P-B-S flick the slides.

To remove the excess D-E-P-C-P-B-S add 200 microliters of the primary antibody solution to each section. After 10 minutes at room temperature, wash the slides with three quick dips in D-E-P-C-P-B-S and flick them once to remove the excess wash solution. Then incubate the samples in 200 microliters of the secondary antibody solution at room temperature after six minutes.

Wash the slides in D-E-P-C-P-B-S as just demonstrated and incubate them in freshly prepared a BC solution for four minutes after another quick D-E-P-C-P-B-S wash mix 196 microliters of freshly prepared DAB with four microliters of RNA inhibitor. Just before adding the solution to the section, allow the DAB to develop for one to two minutes, followed by A-D-E-P-C-P-B-S wash. Then dehydrate the sections with a few seconds incubation in absolute ethanol and flick the slides dry to proceed to LCM.

Next at 50 microliters of lysis buffer from an RNA extraction kit into the collecting tube cap. Then place one slide under the microscope with the sample side facing the cap. Choose the best magnification for the sample and adjust the focus to see the cells of interest.

Then define the cutting area and start cutting with the laser. Collecting the dissected material in the collecting tube cap ly the collected cells for 30 minutes at 42 degrees Celsius. During this time, precondition an RNA purification column from the RNA extraction kit with 250 microliters of conditioning buffer.

Then load 50 microliters of ethanol to the lysed cells on the column and centrifuge the column for two minutes at 16, 000 Gs and room temperature followed by two rinses with wash buffer. Finally, elute the RNA in the minimal recommended volume of 11 microliters of RNA free water keeping two microliters aside to test the quality of the RNA. This rapid immuno labeling procedure allows the visualization of cells of interest while maintaining the experimental RNA integrity.

Depending on the experiment, LCM can be used as just demonstrated to isolate single cells or larger regions of interest. Degraded RNA has a considerable impact on the quality of the downstream analysis. Each sample is thus further processed on a microfluid X based platform bioanalyzer to allow quantification and verification of the integrity of the isolated RNA as just demonstrated.

For example, here, typical bioanalyzer results of both degraded and good quality. RNA samples are shown. Indeed, digital gel and electropherograms demonstrate the importance of using an rase inhibitor in each solution to protect the RNA following this procedure, high quality RNA can be further processed for downstream applications, including RNA sequencing MICROARRAY or Q-R-D-P-C-R, for example, in these graphs, the average enrichment for TH and A A DC gene expression in the micro dissected sample relative to mid-brain sections was determined to be more than 170 fold.

While at attempting this procedure, it’s important to remember work very quickly and with RNAs free material. After watching this video, you should have a good understanding of how to isolate a specific population of cell from any tissue.