Tapping into Stem Cells
Researchers at Life Technologies began to explore the expression profiles of embryonic stem cells a few years ago and discovered distinct miRNAs highly expressed in these cells. This subset of miRNAs associated with pluripotent cells is thought to be important for maintenance of stem cells in their undifferentiated state. This work was enabled by internally developed miRNA profiling tools.
“One of the challenges was using these tools for mesenchymal stem cells because they are not as homogeneous as embryonic stem cells. We had to adapt an alternate approach to identify potential gene targets of the identified miRNAs and validate their role in a particular pathway. So it was a bit more complicated,” explains Uma Lakshmipathy, Ph.D., principal scientist.
The next challenge is to identify specific gene targets of these miRNAs and the effect of miRNA-target interaction on transcriptional networks and signaling pathways. “This information will be critical in identifying the precise role of miRNAs in a particular cellular process,” adds Dr. Lakshmipathy. She adds that another key challenge is to validate all the cell-type specific miRNAs identified so far.
In order to address these challenges, the company has developed miRNA array platforms for analysis of known miRNAs in cells (NCode miRNA multispecies/human array), the IVGN NCode miRNA Microarray kit v3 with novel human miRNA and all known miRNAs in the Sanger miRBase Sequencing Database, a multispecies miRNA kit (v2), and ABI TaqMan Human MicroRNA array.
In addition, there are also reagents for the SOLiD sequencing platform, designed for whole genome analysis and discovery of small noncoding RNA and miRNAs. Potential applications include diagnostics for identification of biomarkers associated with disease, characterization tools to qualify stem cell populations used as therapeutics, as well as embryotoxicity assays to be used in the drug discovery process. “The ultimate goal is to use miRNAs to modulate gene expression to obtain desired cell effects,” summarizes Dr. Lakshmipathy.
Most pancreatic cancers are diagnosed late because they remain asymptomatic until the end stages of disease. Scientists at Asuragen have developed a miRNA-based qRT-PCR assay that differentiates pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis. “There can be masses associated with pancreatitis that are benign, so you don’t want to put the patient through surgery if you don’t have to,” says Anna Schwarzbach, Ph.D., CLIA lab supervisor.
Two differentially expressed miRNAs—miR-196a and miR-217—have shown the ability to distinguish PDAC from chronic pancreatitis. The assay using this miRNA signature was validated using FFPE pancreatic blocks and achieved close to 95% specificity and sensitivity, Dr. Schwarzbach said.
Some preliminary data with fine needle aspirates of PDAC and normal pancreatic tissue demonstrated these two miRNAs could identify PDAC patients. Dr. Schwarzbach says they are now in the process of establishing collaborations to obtain larger numbers of fine-needle aspirates for a larger, multicenter study. This will allow for true assessment of the assay’s clinical and analytical parameters in fine-needle aspirate tissues.
Some advantages to working with miRNAs include the fact that they may potentially have more potent changes in expression upon disease development than mRNAs because there are single miRNAs that can regulate multiple mRNAs. Also, since they are smaller than mRNAs, they are less prone to degradation and are easily recovered from FFPE samples.