This is a highly accurate and cost effective method for understanding gene expression at the single cell level. The main advantage of this technique is that we can fit 96 samples in one batch. This high number of samples allows us to identify cellular subphenotypes with an anatomic specificity.
This method's pipeline can be applied to any biological problem, any system, any tissue, in order to inquire as to the individual cellular response of individual neurons or other cell types to disease or any other disturbance, any function. And then go further and inquire as to whether those cell responses have an anatomical architecture. Remove the brain from the tissue collection box and place onto the cryostat chuck.
Collect 10 micron sections containing the central nucleus of the amygdala or other preferred brain region by thaw mounting 10 micron sections onto plain glass slides. Immediately place the glass slides onto a metal pan resting on dry ice. Put the slides with brain sections into a minus 80 degrees Celsius freezer as soon as possible.
To perform an ethanol and xylene dehydration series, first dip the slides into 75%ethanol for 30 seconds. Immediately after, dip the slides in 95%ethanol for 30 seconds. Then dip the slides into 100%ethanol for 30 seconds.
Finally, dip the slides directly into a second container of 100%ethanol for 30 seconds. Following the ethanol dehydration series, dip the slides into freshly poured xylene for one minute. Immediately after, dip the slides into a second container of xylene for four minutes before drying as described in the text protocol.
Use fluorescence to identify the stained cell type and its nucleus in the region of interest. Choose one cell or multiple cells if doing single cell pooled samples. Mark the cells of interest using laser capture microdissection or LCM software.
Place the LCM cap on top of the slice on the region of interest and melt the LCM cap adhesive over the area of the selected single cell as described in the text protocol. Select the individual cells to be collected for analysis using the LCM software tools. Cells selected must be in the anatomic area of the central nucleus of the amygdala based on the rat brain atlas and the bregma.
Cells should be at least three microns from the adjacent stained nuclei. Then fire the infrared laser to collect the identified single cells. Place the cap in the QC station and view it to ensure that only the desired cells were selected.
If other cells were mistakenly selected, an ultraviolet laser can be used to destroy the unwanted cells while the cap remains in the QC station. Take a photo of the tissue section from where the cell was collected to document its anatomic specificity. Record the distance of the slice from the bregma if appropriate using a rat brain atlas as a reference.
Remove the LCM cap from the QC station and attach the sample extraction device. Then pipette 5.5 microliters of lysis buffer onto the sample. Fit the extraction device onto a 0.5 milliliter microcentrifuge tube and place it on a hot plate at 75 degrees Celsius for 15 minutes.
Spin down the sample and lysis buffer for 30 seconds at low speed and place the collected sample into a minus 80 degrees Celsius freezer. To perform pre-amplification of single cell mRNA for a dynamic array chip, first add five microliters of 5X VILO to each well of a new 96 by 96 PCR plate. Remove LCM single cell samples from minus 80 degrees Celsius and let them thaw briefly.
Following centrifugation at a low speed, add 5.5 microliters of the lysed single cell samples to the PCR plate adding each sample to its own well. Place the PCR plate with the samples and VILO into the thermocycler and heat at 65 degrees Celsius for one and a half minutes. Then spin the plate for one minute at 1, 300 times g and four degrees Celsius and place the plate on ice.
Add 10X cDNA synthesis master mix, T4 Gene 32 Protein, and DNA suspension buffer to each well. Place the PCR plate which is sample plate one into the thermocycler and run as detailed in the text protocol. After the cycle, add nine microliters of Taq polymerase solution to each well in the 96-well PCR plate.
Nine microliters consist of 7.5 microliters of Taq polymerase master mix and 1.5 microliters of the primer pool. Now, place the PCR plate in the thermocycler and run the pre-amplification program detailed in the text protocol. Following the pre-amplification protocol, add six microliters of exonuclease solution to each well in the 96-well PCR plate.
The exonuclease solution consists of 0.6 microliters of 10X exonuclease-one reaction buffer, 1.2 microliters of exonuclease-one, and 4.2 microliters of DNA suspension buffer. Place the PCR plate in the thermocycler and run the program listed in the text protocol. Finally, add 54 microliters of TE buffer to each well of sample plate one.
Spin the PCR plate at 1, 300 times g for five minutes. Store at four degrees Celsius if immediately continuing to the next step. In a new 96-well PCR plate which is sample plate two, add five microliters of DNA binding dye and master mix solution.
Add three microliters of pre-amplified samples from sample plate one into the corresponding wells in sample plate two. Spin the PCR plate at 1, 300 times g and then put the plate on ice. In a new 96-well PCR plate which is assay plate two, add five microliters of assay loading solution to each well.
Assay loading solution consists of 3.75 microliters of 2X GE assay loading reagent and 1.25 microliters of DNA suspension buffer per well. Then add 2.5 microliters of 10 micromolar qPCR primer from assay plate one to its corresponding well in assay plate two. Spin the PCR plate at 1, 300 times g for five minutes.
To load and run the dynamic array chip, first prime the chip with control line fluid. Then place the chip in an IFC controller HX and run the prime 136X script. Add six microliters from sample plate two into the corresponding sample wells in the 96 by 96 dynamic array chip.
Now, add six microliters from assay plate two into the corresponding assay wells in the 96 by 96 dynamic array chip. Use needles to pop any air bubbles in the wells of the 96 by 96 dynamic array chip. Then place the chip into the IFC controller HX and run the load mix 136X script.
Next, remove the chip from the IFC controller HX.Place the 96 by 96 dynamic array chip into a microfluidic RT-qPCR platform and run the GE fast 96 by 96 PCR protocol. The selection of the single cells was validated both visually and molecularly. Visually, cellular morphology was viewed before cell collection.
Shown here are representative images of a slide with hemisected rat forebrain containing the central nucleus of the amygdala. Subsequent images show the selection of single cells and their removal from the tissue for transcriptomic analysis. Molecularly, cell type specific markers demonstrated increased expression in their respective cell type.
Specifically, increased expression of NeuN was observed in neurons, Maf in microglia, and Gfap in astrocytes. The heat map shows the expression of all samples across 40 assayed genes. Rows are tensile pooled samples, the numbers denote the sample clusters and the columns are the genes.
Multivariate analysis methods show that astrocytes in the withdrawal group were the most affected cell type. Based on these data in the context of other studies, astrocytes likely play a key role in inflammation in the central nucleus of the amygdala during opioid withdrawal. The most important thing to remember about this technique is to use proper pipetting to limit the number of air bubbles.
The transcriptional findings from these experiments can be validated using protein measure methods such as immunofluorescence or Western blot on the same tissue.
