Research Techniques

Cancer research would not be possible without a wide variety of research technologies and techniques to make it possible. Some are old technologies that have been improved in the years since their development; others are novel technologies that emerged in the modern day era.

Some of the common laboratory techniques used at the Onco-Sera Research Center are listed below. We also discuss how each technology is applied to our cancer research projects.

Flow cytometry

One of OSRC’s major projects is currently studying pancreatic cancer. Pancreatic cancer is a major challenge for both doctors and researchers because of its aggressiveness. The survival rate for pancreatic cancer in comparison to other types of cancers is much lower and scientists at OSRC are working hard to find ways to improve pancreatic cancer patient outcomes.

This research relies heavily on the use of high-throughput sequencing technologies. However, a pancreatic tumor mass is not necessarily abundant with tumor cells. Only approximately 10 per cent of cells in a pancreatic tumor mass are tumor cells, the mass being mostly composed of blood cells, blood vessels, and fibroblast cells. A high-throughput sequencer acquires information from the most abundant cells in a sample and in pancreatic tumor samples, this is not always the tumor cell. This is a major problem in the progression of research within pancreatic cancer and is solved using a cell sorting technology, called flow cytometry. Flow cytometry makes use of existing knowledge that each type of cell expresses different types of antigens or cell surface markers. By labeling these cell surface markers with specific fluorescently-tagged antibodies, the pancreatic tumor cells from a patient sample can be separated from the other cells that may also be present in the sample.

 

DNA Sequencing

Identification of cancer-causing mutations in DNA is the basis of many of the projects at the OSRC. Working toward identifying mutations in the sequence such as small insertions, deletions or single nucleotide variants (SNVs) using a sequencing machine takes about seven day for one run and produces approximately 300 gigabytes of useful data.

This process first involves preparing the samples to be analyzed. Samples are collected from cancer patients; usually both tumor cells and normal cells are collected for comparison. DNA from each of the samples is extracted and fragmented to a specific size. Adapters are added to the DNA fragments, the DNA denatured (meaning the double stranded fragments of DNA are separated into single strands) and bound to the flow cell via adapter complementary primers that are affixed to the surface of the flow cell. A flow cell is similar to a microscope slide with eight individual lanes etched into the slide where the DNA is attached and reagents flow through. The initial step in high throughput sequencing of a DNA sample is called bridge amplification, where each bound single strand of DNA is replicated to form a cluster of identical DNA fragment molecules. Each lane on the flow cell slide has thousands of clusters, each cluster representing a different DNA fragment. At this point, the flow cell is ready to be sequenced.

The sequences are computationally aligned and compared to the human reference genome to gain position and annotation information. Once all of the clusters of DNA sequence have been arranged in order, the normal and tumor samples from a patient can be compared. Differences between normal and tumor cell DNA allows scientists to identify mutations present only in the tumor sample and to formulate hypotheses surrounding which mutations may be associated with producing the cancer.

Immunohistochemistry – Cell Pathology

At OSRC, there are a variety of different devices that allow scientists to see the cells they are studying. The most common way to visualize cancer cells is with a high quality research microscope.

Tumor samples are prepared in a grid on a traditional glass slide in a process called tissue microarray. Scientists extract a small sample (called a core) the size of a pin from a tumor mass and place it into a wax block. This process is repeated 200 times to make a grid of different tumor samples). The wax block is then sliced ultra-thin and a slice placed onto a glass slide.
The cells are then stained using immunohistochemistry. Labelled antibodies matching a desired antigen are added to the slide. If any of the tumor samples express these antigen proteins, the antibodies will attach to the tumor antigens and stain the tumor. Under a light microscope, the tiny details within each cell become clearly visible. In particular, stained tumour cells become visible and indicate which tumors contain the protein of interest.

Often, a tumor sample will have cells in addition to the tumor cells. Laser capture microdissection technology allows scientists to identify and isolate the tumor cells from the other cells. With this technology, scientists view the tumor slide under a microscope, and select a specific group of tumor cells they are interested in. Using a laser, the area is cut out and moved into a micro-tube to allow for extraction of DNA, RNA, proteins, or other molecules.
Microscopy is used at all stages of cancer research from determining basic molecular structures to determining effectiveness of new treatments. By allowing scientists to visualize the details of cancer cells, microscopy is an essential technology in cancer research.

Other Techniques:

At Onco-Sera our researchers also utilize the classic methodologies of research laboratory practice, namely, High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), Enzyme-linked immunosorbent assays (ELISA), Electrophoresis methods, Horizontal, Vertical, Use of Antibodies, Monoclonal and Polyclonal, Custom Probes, Primers and Oligonucleotides, Restriction Enzymes, Fine Chemicals, Bacterial Identification media and much more.