Every protocol for single cell PCR can be broken down into two steps. In the first step, the cells are isolated by micromanipulation, laser capture microdissection, flow cytometry, or by direct micropipetting. Next, the genetic material is processed by PCR to amplify your sequence of interest.
Here, we’ll go through the different options for isolating your cell.
Note: AmpliGrid is a collector of glass slide-size that will make your single-cell isolation a lot less of a headache. It allows you to check your cell has been successfully transferred thus improving overall results. Laser microdissection is still time consuming however. While this lab-on-a-chip technology allows you process 48 samples in very low volumes, you need a special PCR machine on which to use them.
Task 1: digging for treasure – how to isolate your cells
Applications: Forensic science, reproductive medicine and bacterial cell analysis.
This is the time tested standard way to handle single cells. The equipment consists of a joystick connected to an inverted microscope. The joystick controls the platform of the microscope.
Drawbacks: Although this method is powerful it is also slow and there are quality issues. The former problem is as a result of the time it takes to transfer a cell from the medium in which it is suspended to a tube or microtiter plate well. After the micromanipulator leaves the optical plane where samples can be observed, it isn’t possible to visually ensure that each single cell has been properly transferred to the bottom of a tube or well. This causes problems downstream after PCR amplification as negative results cannot be conclusively said to be negative; there may simply have not been a cell added. This problem can be negated by using an AmpliGrid.
Laser Capture Microdissection (LCM)
This method is fast as methods go. Cells of interest can be isolated from tissues, organisms, cell smears, chromosome preparations, paraffin or frozen embedded samples and small collections of cells. A laser is used to cut a predefined section of tissue after it has been chosen manually, semi-automatically or automatically using the microscope. It is a little like a claw machine picking up a toy (although a lot better at successfully giving you what you want!) The laser cutting width is no more than 1 µm, thus live cells are not damaged by the laser cutting and are viable for cloning and culturing.
Drawbacks: The hardware for this technique is expensive and the speed as which cells can be processed has a lot to do with the experience of the histologist observing the samples.
In terms of speed, this guy gets the medal, sorting about 1 million cells per minute. It can process varying numbers of immuno parameters depending on the machine but 8 is the standard. The success rate in terms of placing cells into a well is mediocre (ranging from 50% to 70%) owing to the charge on the plates triggering a deflection of the cells before they hit the bottom of the well. This can be improved drastically using AmpliGrid slides. Correct placement can be verified by fluorescent microscopy afterwards. This gives you peace of mind when analyzing PCR results as you don’t need to wonder if a negative results was due to poor cell placement.
Pipetting from a Petri Dish
This method brings you back to your micropipette and skips the middleman as you’re sorting the cells directly from your culture. Using CellSorter software, you can look through your sample yourself or get the software to do so for you. This software can automatically detect fluorescent cells. The program generates a map to show you where they are and how best to get them. Using a glass micropipette, you can pick up the cells you’re interested in or, again, this can be done automatically for you. Up to a thousand cells can be picked up in one run. The speed varies depending if you’re picking up one cell (20–24 seconds/cell) or multiple cells (1 cell/second). This technique can be technically challenging and is certainly slower than other methods, but it allows you to tell in real time if your cell has made it to where it should be saving you time later in sifting through wells and ending up with a lot of blanks with which to deal.
Task 2: Getting down to business – amplify your cell’s genetic material
First extract your genetical material by cell lysis. Next, use whichever form of PCR best suits your experiment. If using an AmpliGrid, you will need an AmpliSpeed slide cycler. Two of the most commonly used options are:
- RT-PCR: reverse transcriptase enzyme is used during the PCR to convert RNA to complementary DNA (cDNA). This is used if you have an RNA sample to be amplified.
- qPCR: quantitative PCR allows you to measure the amount of your gene of interest present in your sample in real time using fluorescent dyes or DNA probes.
Which single cell isolation method works best for you?