The overall goal of this procedure is to prepare samples from single cells for transcriptome analysis. This is accomplished by first finding a cell suitable for isolation. The micro pipette is then positioned next to the cell to be isolated.
Next suction is applied to the syringe until the cell is pulled into the tip of the micro pipette. The final step of the procedure is to break the tip of the micro pipette into the collection tube and deject the remaining contents of the micro pipette into the tube. Ultimately, results can be obtained that show differences in mRNA populations between cells through microarray analysis of material amplified using the arna amplification method, also described in the protocol or for other applications such as PCR or next generation sequencing.
Although this method can provide insight and the differences between single cells, it can also be applied to subcellular compartments such as dendrites or classes of dendrites, and provide important information into dendrite function. Demonstrating the procedure will be Jennifer Singh, a postdoc in my lab Prior to the start of this protocol, primary neurons from embryonic day 18, rat Hippo Campi were plated onto 12 millimeter cover slips in a 35 millimeter dish and maintained as described in the written protocol. To prepare pipettes for harvest, use a micro pipette puller to pull autoclave glass pipettes to a diameter appropriate for electrophysiology.
Using the manufacturer's preset program, the bore size is not critical since the tip will be broken prior to cell collection. Carefully store the pipettes in a dust-free environment, such as a micro pipette storage jar, where the tips will remain intact when ready to harvest cells. Prepare the desired number of 1.7 milliliter einor tubes by adding two microliters of PBS for storage of the harvested material.
Remove 1 35 millimeter dish of cover slips from the incubator in a tissue culture hood. Quickly transfer one cover slip from this dish to the lid of a new 35 millimeter dish containing HBSS. It is critical to return cover slips that are not being manipulated to the incubator as the cells become unhealthy.
If left out for extended periods of time. An inverted light microscope with a 40 x objective fitted with a micro manipulator is necessary for cell harvesting. Use a pipette holder with flexible tubing leading away from the holder, affix the tubing to a stable surface to prevent unwanted movement of the pipette during collection.
At the end of the tubing, insert a needle so that a one milliliter syringe with a lure lock connection can be attached and changed in between users. Prepare the micro pipette by breaking the tip in a controlled fashion by holding a Kim wipe taut between the fingers of one hand and very gently brushing the tip of the pipette across the paper one to two times. The opening should be approximately 75 to 100%of the target cell size.
Secure the micro pipette in the micro pipette holder and affix the micro pipette holder to the insert on the micro manipulators. Finally, set the 40 x objective. Place the dish on the microscope stage and find a region of cells suitable for harvest.
In the case of primary neuronal cultures, the cell should be relatively isolated from its neighbors. To prevent harvest of surrounding cells and or processes, place the cell to be harvested in the center of the field of view. Use the micro manipulator to advance the micro pipette toward the solution in the dish into the light path.
Lower the pipette tip into the solution. Looking through the eyepiece, the pipette should appear as a shadow in the field of view. Apply positive pressure from this point on by blowing lightly through the syringe well above the plane of cells.
Adjust the position of the pipette tip until it is within the field of view and focus on the pipette tip using the course focusing knobs. At this point, assess whether the bore size of the pipette is too large or too small. If the bore size is incorrect, replace with another micro pipette until the correct bore size is found To prevent pipette breakage in the collection region.
Use the course focus to advance the focal plane before advancing the pipette tip towards the cells using the microm manipulators. As the cells become closer, use the fine focus until both the cells and the pipette tip are in focus. Stop applying positive pressure before the plaintiff cells is reached to avoid blowing them off of the cover slip.
Position the pipette tip with the micro manipulators so that it is touching the cell soma to be harvested. Using the syringe, apply gentle suction by mouth until the cell enters the pipette tip. If the cell is not entering the pipette, gently move the tip closer to the cell and downward until it lifts from the cover slip.
Use the micro manipulator to immediately move the pipette up and out of the solution. Remove the pipette from the holder. Hold the prepared 1.7 milliliter einor tube in one hand, and gently break the tip of the pipette on the side of the tube toward the bottom.
Aiming for the 0.1 milliliter mark with the broken tip centered inside the tube, insert a needle attached to a one milliliter syringe into the top opening of the pipette. Rapidly depress the plunger, forcing the solution in the pipette to spray out into the tube. Take care not to touch the tip to any liquid on the sides of the tube.
As capillary action will bring the liquid back into the pipette, the cell should remain in the very tip of the pipette, and this should be adequate to properly expel the cell. Quickly spin down the contents of the tube using a desktop micro fuge and freeze on dry ice or place on ice for further processing or for other applications such as PCR or next generation sequencing. Shown here is an example of a successful harvest of an isolated neuron.
To begin, a relatively low density region of cells was selected. The pipette tip was then advanced toward the desired cell. Here the field of view is shown after the cell had been harvested.
Note that the surrounding processes remain on the cover slip. Conversely, this figure demonstrates two images of pipette tips, which are an inappropriate size for effective harvesting. This tip will lead to incomplete harvest while the tip shown here will result in harvest of surrounding milieu.
Following harvest, and A RNA amplification analysis, using a bioanalyzer is recommended to examine the distribution and quantity of amplified RNA. A successful amplification from single cell material will yield total amounts in the low micrograms and will have a distribution that is smooth and wide Once mastered, this technique can be completed in minutes if performed properly while performing this procedure, it's important to use RNA free technique.
