The overall goal of this procedure is to isolate single cells for microarray based transcriptome analysis. This is accomplished by first dissecting the retina from the surrounding tissue and then dissociating it into single cells. The second step is to harvest single cells and deposit them into PCR tubes containing lysis buffer.
Then the cells are lysed and the CDNA is amplified using reverse transcription, followed by polymerase chain reaction. Ultimately, hybridization of the single cell CD NA on an A iMatrix microarray is used to reveal the mRNAs expressed in a single cell. The main advantage of profiling the gene expression in single cells versus using whole tissue approaches is that single cell isolation allows you to gain access to very small populations of cells that would comprise the entire tissue.
These cells could be anything from different types of neurons to ling small populations of stem cells. What the single cell technique allows us to do for these cells is to identify both cell types, specific markers for these small populations of cells, and identify gene networks that are expressed in these extremely rare subsets of cells. Additionally, the single cell profiling technique allows us to identify the gene networks that are transiently expressed during very dynamic developmental transitions in very small subsets of cells.
This method can help to answer key questions in the field of developmental genetics, such as when progenitor cells begin to express gene networks associated with differentiation and how those progenitor cells differ from one another. The implications of this technique extend towards cellular therapies for neurodegenerative diseases such as glaucoma, because by elucidating the key networks in the development of individual cells we're one step closer to being able to generate those cells for use in cellular replacement therapy. Begin this procedure by placing the mouse retina in a dish filled with PBS.
Next, remove the vitreous and the lens. Then remove all of the retinal pigment epithelium. Incubate the retina in a 1.5 milliliter micro centrifuge tube at 37 degrees Celsius for 10 minutes.
Afterward, gently tap the tube to dislodge any settled cells. Then tri rate it gently, 10 to 20 times with a P 1000 pipetter. If large clumps of tissue persist, incubate it for an additional 10 minutes at 37 degrees Celsius and ret, refrigerate it 10 to 20 times.
Next, add five microliters of dna, one at 10 units per microliter to the retina, and incubate it for five minutes. At room temperature trier, rate it gently with the P 1000 pipetter again for another 10 to 20 times afterwards. Centrifuge it at 3000 RPM for three minutes.
Then remove the supernatant and leave 100 to 200 microliters of liquid at the bottom. Tap the tube to dislodge the pellet. Next, add one milliliter of HBSS and resuspend the pellet with gentle tation.
Centrifuge it at 3000 RPM for three minutes. When it is done, carefully remove the supernatant. Then resus, suspend it in 450 microliters of PBS containing 0.1%BSA in the following step, prepare two six centimeter dishes containing five milliliters of PBS supplemented with 0.1%BSA.
Next plate, the dissociated cells onto one dish. Allow the cells to settle for at least five minutes. After that, place the dish of dissociated cells under an inverted microscope.
Use a fine tipped pipette with an aspirator tube to harvest a single cell. Then place the micro pipette close to the cell and it will readily enter the micro pipette via a capillary action. Subsequently expel the cell onto a second dish and ensure that only one cell is harvested for gene expression profiling.
This is done by either removing extraneous cells or moving the cell of interest to a different location with a new micro pipette. When an individual cell is completely isolated from the neighboring cells, use a new micro pipette to aspirate the cell of interest. Next, expel it directly into a 0.2 milliliter PCR tube containing 4.5 microliters cell lysis buffer.
Spin the sample briefly in a micro centrifuge to immerse the cell in lysis buffer. Do this spinning after every fifth cell and then again at the end of collection. Remember to keep the tube on ice for the duration of the single cell isolation and collect the single cells within a two hour window post dissociation for optimal results in this procedure, briefly spin the sample in a micro centrifuge to ensure that the single cell is immersed in the cell lysis buffer.
To promote cell lysis, incubate the sample at 70 degrees Celsius for 90 seconds in a thermocycler. Then immediately place the tube back on ice for two minutes. To perform the reverse transcription, add 0.33 microliters.
Superscript three at 200 units per microliter 0.05 microliters inhibitor at 40 units per microliter and 0.07 microliters. T four gene 32 protein to the tube. Incubate the reaction mixture for 50 minutes at 50 degrees Celsius in a thermocycler after 50 minutes, inactivate the enzyme at 70 degrees Celsius for another 15 minutes.
When that is done, place the tube back on ice. To remove the free primer add 0.1 microliters 10 x exonuclease buffer, 0.8 microliters, D H2O and 0.1 microliters. Exonuclease, one at 20 units per microliter to the tube incubated at 37 degrees Celsius for 30 minutes in a thermocycler.
After 30 minutes, inactivate the enzyme by incubating the reaction mixture at 80 degrees Celsius for 25 minutes. At the end, place the tube back on ice. Next, add six microliters of the tailing reaction mixture to the sample.
Incubate it in the thermocycler at 37 degrees Celsius for 20 minutes, 70 degrees Celsius for 10 minutes, and then keep it at four degrees Celsius. Subsequently, add 10 microliters of the tailed sample to the PCR reaction mixture. Perform the second strand synthesis and PCR amplification to label CDNA.
In order to obtain a robust hybridization on a iMatrix microarrays first fragment the CD NA by adding 10 to 20 microliters of the single cell CD NA to eight microliters one x one for all buffer. Then add one microliter of this diluted DNA one to an 80 microliter reaction solution. Next, add 20 microliters, five XTDT buffer, 2.5 microliters biotin, and six D-D-A-T-P and one microliter diluted TDT to it.
Next, add 20 microliters, five XTDT buffer, 2.5 microliters, biotin, and six D-D-A-T-P and one microliter TDT to it. Incubate the mixture for 90 minutes at 37 degrees Celsius. Then five minutes at 65 degrees Celsius.
Subsequently store it at minus 20 degrees Celsius or hybridize immediately to an a iMatrix microarray. Here is a representative example of single cell CD NA smears and gene specific PCRs lanes A-F-G-N-H contain robust CD NA smears while lanes B, C, D, and E are considerably less robust. CDNAs were isolated from fluorescent retinal ganglion cells, and were then tested using PCR primers specific for the retinal ganglion cell markers, BRN three B and PAC six, gene specific PCR for the photoreceptor marker.
CRX was used to determine the level of contamination in the single cell. CDNAs and subsets of retinal ganglion cells were identified by screening for the presence of the gene tachy kinin one. The microarray results for selected genes expressed in 22 distinct single cells are shown in a heat map format.
The intensities from the microarray signals have been scaled to correspond to the intensity of the red color, while black indicates the absence of signal on the microarray. Following this procedure, additional methods such as QPCR can be used to confirm the gene expression pattern seen in microray. Results in C hybridization can also be used to answer additional questions you may have about the locations of cells that express particular genes.
After watching this video, you should have a pretty good idea of how to dissociate retinas, isolate single cells, and generate CDNA from those single cells. For hybridization on microarray.
