This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.
This method can help answer key questions in neuroscience research, such as characterizing neuro population or identifying sources and targets of transmission systems. The main advantage of this technique is that it allows to determine the expression profile of a pre-selected set of genes from a single cell quickly and easily. So this method can provide insight into the random diversity mouse.
It can also be applied to other cell types, such as astrocytes and in many animal models. This demonstration of this method is critical as the arresting and aspiring steps can be difficult to learn because the data needs a quantity of cytoplasm to be analyzed. Before sectioning, prepare a dissection kit containing surgical scissors, fine iris scissors, two spatulas, forceps, a disc of paper filter, and cyanoacrylate glue.
Then saturate the ice cold cutting solution with carbogyn and cool down the cutting chamber at negative 20 degrees Celsius. Next, remove the animal's scalp and open the skull. Extract the brain carefully and place it into a small beaker filled with carbogenated ice cold cutting solution.
Afterward, remove the cutting chamber from negative 20 degrees Celsius and remove the moisture with a paper towel. Carefully dissect the brain to isolate the region of interest. Glue it on to the cutting chamber and add the carboginated, ice cold cutting solution.
Using a vibratone, section 300 micrometer thick slices. Transfer the slices to a resting chamber filled with oxygenated cutting solution at room temperature and allow them to recover for at least 30 minutes. For single cell RT-PCR and the targeted cell characterization, limit whole cell recording to no longer than 20 minutes in order to preserve the mRNAse.
At the end of the recording prepare a 500 microliter PCR tube filled with two microliters of 5xRT mix and 0.5 microliters of 20xDTT. Spin it and store it on ice. Then harvest the cell's cytoplasm by applying a gentle negative pressure.
Monitor and control the cell's content from getting into the pipette while maintaining a tight seal and avoid collecting the nucleus if some genes are considered. In the case of gene, it is essential to avoid the collection of the nucleus and to include a set of primers aimed at amplifying gDNA to prep for general contamination. If the nucleus is getting close to the tip of the pipette, release the negative pressure and move the pipette away.
Ensure that the tight seal is preserved and restart the collection of the cytoplasm at the new pipette location. Next, withdraw the pipette gently to form an outside out patch to limit contamination by the extracellular debris and to favor the closure of the cell membrane for subsequent histo-chemical analysis. Keep in mind that nubs, micro-manipulators, computer keyboard or computer mouse, are proton charged sources of rnase contamination.
If they have been touched during the recording, change the gloves. Subsequently, attach the pipette to the expeller, and expel its content into the PCR tube by applying positive pressure. Break the tip of the pipette into the PCR tube to help the collection of its content.
Then, briefly centrifuge the tube add 0.5 microliters of RNAse inhibitor and 0.5 microlitres of RTAse. Mix gently, centrifuge again, and incubate overnight at 37 degrees Celsius. The next day, spin the tube, and store it at negative 80 degrees Celsius for up to several months.
Until PCR analysis and performing the first amplication step. To validate PCR, mix and dilute a primer pair, add one micromolar with the pipette dedicated to the single cell RTPCR. Set the thermo cycler for three minutes at 95 degrees Celsius, run 40 cycles followed by a final elongation step at 72 degrees Celsius for five minutes.
To minimize the formation of primer dimers, perform a hot start by placing the PCR tubes in the thermo cycler preheated at 95 degrees Celsius. After 30 seconds, quickly expel 20 microliters of the primer mix on top of the oil. Once the primer pair has been validated, test it with the multiplex protocol.
Mix and dilute the external primer together at one micromolar. Store the multiplex primer mix at negative 20 degrees Celsius for up to several weeks. Next, prepare a pre-mix and place it on ice.
Flick the PCR tube and spin it and add two drops of mineral oil. After three minutes at 95 degrees Celsius, run 20 PCR cycles. Perform a hot start as described with 20 microlitres of multiplex primer mix.
Then, prepare a number of PCR tubes identical to the number of genes being analyzed. Prepare a pre mix using one microliter of the first PCR product per gene as template. Adjust all the volumes according to the number of genes to analyze and consider using 10%of extra volume to compensate for pipetting errors.
Shake gently, spin the tube and dispatch 80 microliters of premix into each PCR tube. Add two drops of mineral oil per tube without touching the wall or the cap of the tubes. Run 35 PCR cycles then perform a hot start by expelling 20 microliters of internal primer mix.
Analyze the second PCR projects by aragose gel electrophoresis. A layer five pyramidal neuron was visually identified by it's large soma and a prominent apical dendrite. Pull-cell recording revealed typical electro-physiological properties of layer five regular spiking neurons with a low input resistance, long lasting action potentials, and pronounced spike-frequency adaptation.
The molecular analysis of this neuron revealed the expression of vGluT1, confirming its glutamitergic phenotype. This neuron also expressed somatostatin and the ATP1-alpha-1 and three sub-units of the sodium-potassium ATPAs. Biocytin labeling of the recorded neurons, confirmed a pyramidal morphology.
As expected from the glutamitergic neurons, the molecular analysis of 26 layer-five pyramidal cells, revealed the expression of vGluT1 but neither of the two GADs. Once technique can be done within two days, if it's preformed properly. While attempting this procedure, it's important to remember to be very careful to avoid contaminations.
After watching this video, you should have a good understanding of how to probe the expression of 10s of genes simultaneously from single cells characterized by patch clumps according to brain slices, after cytoplasm harvesting, reverse transcription, and hot-start PCRs. Don't forget that working with Ethidium bromide can be extremely hazardous and precautions such as wearing gloves and lab coats should always be taken while performing this procedure.
