The goals of this presentation are to first illustrate the isolation of viable adult mouse cardiomyocytes and then demonstrate two single cell gene expression analysis methods. After dissecting the heart from a mouse, the heart is perfused with a collagenase digestion solution to obtain single cells. For quantitative PCR of single cells, the purified cells are directly subjected to QPCR assays.
The isolated cardiomyocytes can also be used for microarray analysis. Microarray analysis of single cells involves mRNA extraction, whole transcriptome amplification via an plex library, followed by limited amplification, and finally, labeling and hybridization to a micro array chip. The final step of both procedures is to ensure high quality data.
These procedures demonstrate that robust gene expression analysis is possible from individually isolated adult cardiomyocytes. The advantage of this method over existing methods, such as gene expression profiling of whole tissue homogenate, is that single cell gene expression allows you to gain information about the expression within a single cell, as well as the variability in expression between cells. Begin with a prepared isolation system, including required solutions and buffers terminally sedate, a mouse with an IP injection of 0.25 milliliters, sodium pentobarbital solution, and ensure the mouse is unconscious and unresponsive to pain stimuli.
Do not use carbon dioxide or cervical dislocation to euthanize the mouse before the mouse stops. Breathing carefully cut. Open the thoracic cavity along the thoracic margin through the diaphragm.
Then cut superiorly upwards and reflect the anterior rib cage. Gently suck the heart into a precut plastic pipette and excise the heart from the thorax. Drop the heart into ice cold digestion.
Buffer A and rinse the blood away after 15 to 45 seconds, perform a second brief rinse in, buffer A, and then transfer the heart into the Petri dish with buffer A and place it under a dissection microscope. Carefully isolate the aorta and its branches. Trim the aorta to just inferior to the last branch.
Fit the cannula tip into the aorta above the coronary arteries and ligated in place with four oh or six oh suture. Begin the perfusion using digestion buffer A.The outflow will go from red to clear as blood is flushed from the heart. The amount of time you take between sacking the animal and hooking up the heart to the profusion rig is critical.
Ideally, you wanna keep this under five to 10 minutes. Also, the temperature of the fluid running through the profusion rig needs to be kept at 37 degrees Celsius. If it's too hot or it's too cold, this will destroy the tissue.
Once the blood is washed from the heart, switch to using digestion. Buffer b perfuse the heart for two to three minutes until the heart starts losing its shape and begins to look rounded. The myocardium should appear lighter as the perfusion proceeds now cut the ventricles off and mince them into smaller pieces.
Place the ventricle tissue into a 50 milliliter conical tube containing 15 milliliters of warm collection buffer. Mix the tissue in the buffer gently with a plastic pipette to make a single cell cardiomyocyte mixture. After one minute, transfer the mixture to a new tube and let settle after the pellet is formed.
Discard the super named and resus. Suspend the pelleted cells in five milliliters of the neutralization wash buffer. The cells are now ready to be selected for single cell analysis.
Dilute the cells down in a small Petri dish so that they may be selected individually. Under a microscope, select healthy individual cells using a pulled pipette attached to an airlock. When picking cells, be sure to avoid contamination and minimize the capture volume to less than two microliters.
Place the individual cells on the bottom of individual PCR tubes and freeze them immediately on dry ice. Then store the cells at minus 80 degrees Celsius to begin resuspend all primer pairs for the genes of interest at 100 micromolar and one XDNA suspension buffer, combine and dilute the primer pairs to 20 micromolar. Lastly, dilute all primer pairs into one solution with a final concentration of 200 nano molar.
With the primer solutions prepared, assemble the master mix immediately remove frozen cells from the freezer and centrifuge them at maximum force for 30 seconds. Then add nine microliters of the master mix to each tube and proceed with the PCR. Remove the tubes from the PCR machine and add four microliters of exos it to each reaction.
Then run each reaction for 15 minutes at 37 degrees Celsius, followed by 15 minutes at 80 degrees Celsius. Finish by holding them at four degrees Celsius. Next, dilute the samples one to five with DNA suspension buffer.
The samples are now ready for quantitative PCR analysis. We now use this material and fluid IMS BIOMARK system using the 48 point 48 or 96 point 96 gene expression arrays. It is possible to use more traditional QPCR formats such as 96 well and 3 84.
Well format, however, this restricts the number of assays which can be performed from a single cell. This process utilizes Sigma's WTA two kit, which can amplify double stranded CDNA from both pellets and single cells. The kit first disrupts the membrane of the single cells and then amplifies the messages in two rounds.
After performing the library prep step, add one fifth of the library sample to 70 microliters of the WTA two amplification master mix and amplify the samples for 25 cycles using the kits parameters. Next, purify the samples using kayak quick PCR purification kit and process them on Cain's kayak cube. When finished, concentrate the purified samples to five microliters using a speed vacuum and use the WTA two kit to put the samples through 17 additional cycles of amplification using the same parameters as the previous amplification.
Purify the samples as done before and check their quality using a NanoDrop spectrophotometer and agilent's bioanalyzer DNA 7, 500 kit. Now label two micrograms of double stranded CD NA from each amplified cell sample using nimble gen's one color labeling kit hybridized five micrograms of CY three labeled sample to a NimbleGen gene expression array after the hybridization wash and dry the slide so they can be imaged. Using a molecular device's gene picks 4, 200 a scanner with the settings calibrated to 100 POW and 300 to 350 PMT.
Analyze the gene chips, then generate pair files for each array using nimble gen's, nimble scan software, and proceed with data analysis. A successfully profused heart yielded a high percentage of healthy heart cells that retained their typical rectangular morphology. If the profusion had not worked well, many cells would've died.
Cells were amplified by the WTA two method and the quality of the end products was tested. The sample showed robust amplification on both the nano drop spec spectometer reading, as well as the electropherogram readout from the bioanalyzer chip. In summary, many genes were robustly expressed in the cardiomyocytes.
However, not all assays are equally reliable as seen in this heat map of peak melt temperatures from a subset of nano fluid at QPCR data. Each row of this map represents one single cell sample in 43 separate QPCR assays. Some assays are quite variable and probably less reliable than those that had higher PCR specificity.
When completing this procedure, it's important to remember that you need to obtain quality cells upfront. This will ensure the quality of your gene expression profiling downstream. Once mastered, the single cell isolation procedure can be completed in under two and a half hours, and the gene expression profiling from these cells can be completed in under two days.
