Laser Microdissection Combined with AmpliGrid/LOC Devices: High-Throughput for Efficient Screening

February 27, 2012

LOC technology enables complex biochemical and physical processes to run in a small space using a minimum of chemicals. Generally, LOC is a minimal device in the form of a portable quick test. This frequently capillary-based microfluidic system is normally accommodated on a substrate no larger than a chip card. The AmpliGrid described here is not a microfluidic, capillary system, but a chip with hydrophilic and hydrophobic spots on which the reaction takes place.

AmpliGrid/LOC devices

The AmpliGrid is a special form of LOC (Lab on a chip). This non-capillary system can serve as a collector for specimen material isolated by various laser microdissection devices. Here, we take a closer look at the Leica LMD6500/7000 system.

The dimensions of an AmpliGrid are those of a standard glass slide. The special feature of this slide is its 48 reaction compartments with hydrophilic and hydrophobic areas on which, for example, dissected single cells are captured. This makes it a HTS (high throughput screening) system capable of processing 48 samples on a single slide in parallel. Systems such as these ensure high sample processing rates for PCR analysis or RT-PCR (reverse transcriptase PCR) approaches.

This chemically structured slide serves as an amplification platform for ultra-low-volume applications. When using a Leica LMD6500/7000, fluid does not necessarily have to be applied to the reaction spots prior to dissection, as is the case with instruments that use the catapult technique. The provision of a buffer solution is not essential, as dissected single cells fall onto the AmpliGrid by the force of gravity and can therefore be collected "dry". Dry collection also has the advantage of easier visualization of the dissectate after laser microdissection.

The AmpliGrid is easily used on special PCR machines. After single-cell dissection, PCR or RT-PCR using ultra-low-volume amplification technology is immediately possible on the 48 reaction spots of the AmpliGrid. The drops (usually 1 µl buffer) of a reaction mixture applied to each spot are held in shape by a micellar surface created by hydrophilic and hydrophobic interactions. The applied drop plus the dissectate are then additionally coated in oil.

The addition of a reaction mixture and/or the application of heat during PCR automatically induces cell lysis, releasing the genetic material of the dissectate.

Extremely small samples with a volume from the picoliter to the microliter range can be directly and thoroughly analyzed on the well of an AmpliGrid. As dissected single cells contain small amounts of DNA or RNA, this is a key advantage. Even the tiniest loss of material soon falsifies results when dealing with such small amounts. Using an AmpliGrid as collector after laser microdissection with the Leica LMD6500/7000, the otherwise necessary purification, washing and transfer steps can be omitted and the genetic material is completely retained. Using the Leica LMD6500/7000, cells no longer have to be individually labeled – this can be done fully automatically with the AVC software.

Fig. 1: The easy to use Leica LMD software interface enables the user to collect dissected cells directly to the spots of the AmpliGrid Slide.

Fig. 1a: Investigate your collection spot from the LOC device (AmpliGrid) before cutting.
Fig. 1b: Dissect your area of interest and investigate your result.
Fig. 1c: Here you can easily visualize the round dissectate in spot A3 after laser microdissection.

Applications of laser microdissection in combination with an AmpliGrid

LOC technology makes it possible to examine the DNA or the expression of many different genes at the same time on the same chip. The single cells that are to be examined, such as neurons, cancer cells, cells from cultures or distinct areas of tissue, or differently treated cell cultures, are first separated from the surrounding tissue by laser microdissection. An AmpliGrid captures the dissected cells, and PCR or RT-PCR analysis can immediately follow. The AmpliGrid collector is excellent for examining DNA and RNA, as 48 different cells or controls can be accommodated on the same chip.

Fig. 1: A) Dissection of single cells by LMD. B) Gravitation-related amplification technique of single cells on only one of the 48 reacting locations of the AmpliGrid. C) By Ultra Low amplification technique a PCR or RT-PCR is possible directly to the reacting locations of the AmpliGrid, hereby the genetic material is made accessible for further procedures. D) Cutting of the AmpliGrid: Detail view of reacting locations with hydrophilic and hydrophobic areas.

DNA analysis (epigenetics) as an application area in cancer research

In cancer research, the laser microdissection/AmpliGrid combination is a successful method for selective analysis of DNA from cancer calls and unaffected cells. Epigenetic analysis of single cells is a field of research, as the cellular heterogeneity of methylation patterns of cancer cells differs from those of healthy cells. Even single cells of the same organ exhibit different methylation patterns depending on the function of a gene, stage of the disease, exposition to environmental factors or stochastic fluctuations. Epigenetic analysis can be performed by using a restriction enzyme-based single-cell methylation assay (RSMA), i.e. a methylation-sensitive PCR analysis technique: To carry out detailed tumor analysis, for instance, defined areas or single cells can be dissected by laser and directed onto an AmpliGrid for DNA amplification through direct PCR. In this case, the AmpliGrid enables high-throughput screening (HTS), i.e. the examination of 48 cells on a single chip. After amplification, the genetic material can be subjected to further techniques such as sequencing, bisulfate modification, epigenetic paging and, with regard to mutations, for example, further qPCR [1].

This method is of course transferable from cancer cells to other cell types (particularly neurons).

RNA applications

Laser microdissection combined with LOC is also a useful method for analyzing RNA in neurosciences, for example. In dopamine research, for instance, laser microdissection is used to isolate single cells in order to detect differences in gene expression in connection with a disease. The examination of single cells in healthy and diseased tissue is necessary in cases of progressive, selective cell loss as with Parkinson’s Disease. Here, laser microdissection enables individually affected cells to be isolated from the substantia nigra of Parkinson patients and compared with unaffected, healthy control tissue. After the cells have been dissected, they can be collected on an AmpliGrid and subjected to various downstream techniques; the AmpliGrid offers simultaneous RT(reverse transcriptase) of 48 samples.

This method is also readily transferable to other cell types [1].