Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

This procedure details the use of laser capture micro dissection to isolate neuronal layers from the olfactory epithelium tissue, and also outlines some downstream gene expression analyses. Start with a nasal biopsy to obtain the olfactory epithelium tissue. Then perform laser capture microdissection to isolate the neuronal layers.

Next isolate RNA from the samples. Reverse transcribe for CDNA and analyze for gene expression by QPCR. Ultimately, laser capture microdissection is used to show that an enriched population of neurons can be isolated from olfactory epithelium tissue and applied to numerous downstream applications.

The main advantage of this technique over existing metals like neuronal cell cultures, is that it allows us to study both state and dynamic changes in a neuronal context. We first had the idea for this project when we discovered the potential of combining laser capture micro dissection with olfactory biopsies to obtain enriched neuronal populations. This method was established and demonstrated by TDA at all.

We expanded on this methodology and combined downstream Q-R-T-P-C-R analysis in patients and controls demonstrating the procedure will be Chen Chung, a technician from our team at Johns Hopkins University. The following procedures will be filmed at the Johns Hopkins facilities Immediately prior to micro dissection. Let frozen slides thaw for 10 seconds, then dehydrate the tissue using an ethanol series using the times and volumes indicated in the text protocol.

Turn on the microscope for photo activated localization. Microscopy set the conditions for 10 x magnification as energy of 82, focus of 74 and speed of 25 to 30 for 20 x magnification. Set the energy to 63, focus to 67 and speed to five to six.

Now visually identify the neurons using 20 x or 10 x magnification. Distinguish the thin surface olfactory neuron layer from the underlying submucosa under a microscope without the use of staining procedure. Observe the neurons as a dense columnar appearance using the pencil function.

Trace around the neuronal layer, trace slightly away from the neurons to prevent burning of the neurons from the laser to obtain high quality RNA. Then initiate the laser guided cutting. Pick up the dissected sections using fine tip micro dissection forceps, and transfer the tissue to a micro tube containing 100 microliters of lysis buffer on ice.

Repeat dissections for all neuronal sections obtained from a single sample within 50 minutes of total time on completion of the dissection, immediately vortex the sample lysates and keep on dry ice. Then store the samples at negative 80 degrees Celsius for total RNA isolation. Thaw the sample lysates on ice.

Carry out total RNA isolation with the RN aqueous micro kit with DNAs one inactivation by following the manufacturer's protocol with no modifications. Elute in a total volume of 20 microliters. Using the bioanalyzer, measure the RNA concentration and evaluate RNA quality with RNA integrity number.

Select samples that meet the quality control criteria for CDNA synthesis synthesize CDNA using a validated commercial kit for each reaction tube. ANIL 0.1 to 1.0 nanograms of RNA in a total volume of eight microliters of RNAs free water, one microliter of oligo DT primer and one microliter of 10 millimolar DNTP Mix. Gently vortex the samples centrifuge and anil at 65 degrees Celsius for five minutes.

After the reaction is completed, immediately place the samples on ice. Prepare the master mix solution as indicated. Then add 9.5 microliters of master mix and 0.5 microliters of commercially prepared reverse transcriptase to each sample tube to the control tube at 0.5 microliters of RNAs free water.

Instead of RT enzyme, run an RT reaction with the first incubation at 50 degrees Celsius for 50 minutes. Then 85 degrees Celsius for five minutes. And a final four degrees Celsius hold.

Dilute all the samples five fold with water and aliquot to determine neuronal enrichment of olfactory marker protein expression. Prepare the PCR master mix solutions using G-A-P-D-H internal control to each well of the PCR plate. Add seven microliters of master mix and three microliters of CDNA.

Then centrifuge the plate for five minutes at 300 gs. Run the real-time PCR reaction using the default thermal cycling conditions specified for the commercial super mix. Analyze the expression results for fold enrichment.

Select the samples that show neuronal enrichment of twofold or more. Next, design a real-time PCR for the expression of genes of interest using a FAM labeled probe of interest and a vic labeled G-A-P-D-H. Set up a PCR plate with eight microliters of master mix in each well and two microliters of CDNA centrifuge the plate for five minutes.

At 300 gs. Run the reaction using the default thermal cycling conditions as per the gene expression master mix protocol provided by the manufacturer. Now analyze the expression results.

This experiment used laser capture, micro dissection and R-T-P-C-R to measure the effect of lithium on the expression levels of glycogen synthase kinase three beta in olfactory epithelium in these two patients with bipolar disorder expression analyses before and after lithium treatment in these two BD patients indicate a decrease in GSK three beta expression while control subjects showed no significant change After its development. This technique paved the way for researchers in the field of neuropsychiatry to explore molecular changes in patient neuronal cells. After watching this video, you should have a good understanding of how to use your laser capture microdissection to isolate an enrich population of neurons, which can then be used for downstream gene expression analysis.