| Amanda Dixon-McIver is finishing her Ph.D. under the supervision of Dr. Silvana Debernardi, in the Medical Oncology Laboratory headed by Prof. Bryan Young in the Institute of Cancer, Barts Hospital, in London, UK. The group has performed microRNA profiling, and Amanda is currently using LNA™ technology for in situ hybridization and microRNA knockdown.
1. What is the current research going on in your lab? Our lab is interested in investigating the role of microRNAs in haematological malignancies, in particular acute myeloid leukaemia (AML). Previously, we have shown that miR-181a expression is strongly correlated with the AML morphological sub-type and with the expression of genes identified through sequence analysis as potential interaction targets 1 . We are currently investigating the functional role of microRNAs in AML, by examining the effect of knocking down particular microRNAs.
2. What is your previous experience in microRNA knockdown? We have not had any prior experience with microRNA knockdown.
3. Why did you choose to use the miRCURY LNA™ microRNA Knockdown Probes from Exiqon? We chose to use miRCURY LNA™ knockdown probes as we had previous experience with using LNA™ probes for in situ hybridization with great success 2 ™ technology itself was the best available for our application. The LNA™ knockdown probes are also fairly easy to deliver into cells, and the published data available 3 suggest that LNA ™ knockdown is very effective. As a negative control for knockdown experiments, we use the scrambled knockdown probe available from Exiqon. It was useful to be able to obtain LNA ™ knockdown probes with a fluorescent label, to allow visual confirmation that electroporation had been successful. We were also able to counterstain the cells with DAPI to visualize the nucleus.
4. In your opinion what are the main advantages to using the miRCURY LNA™ microRNA Knockdown Probes compared to other types of microRNA inhibitors? The main advantage is that it is fairly easy to get the probe into the cell and the published data available suggested that it is very effective.
5. Why did you choose to use electroporation to deliver the LNA knockdown probes into this cell type, and how difficult was it to set up and optimize the conditions? The cell line that we used for this study is Molt4, an acute lymphoblastic leukaemia suspension cell line. We chose to use electroporation to deliver the LNA ™ knockdown probe, as we have found electroporation to be the most effective method for delivery into haematopoietic suspension cell lines in our lab. Electroporation of small RNA molecules can be performed using a square wave pulse electroporator, which results in minimal cell death when the electroporation conditions have been optimized.
6. Currently, what is the biggest challenge in your miRNA research? The biggest challenge in our microRNA research is confirming that the knockdown has been successful. We are currently measuring microRNA knockdown by qRT-PCR, however this may not be the most appropriate method so we are investigating other techniques. We are also investigating an interesting cellular phenotype that we have observed when knocking down our microRNA of interest.
1 Debernardi et al. , MicroRNA miR-181a correlates with morphological sub-class of acute myeloid leukaemia and the expression of its target genes in global genome-wide analysis. Leukaemia (2007) 21, 912 – 916.
2 Dixon-McIver A. et al . PLoS ONE . 2008 May 14; 3 ( 5 ): e2141 .Distinctive patterns of microRNA expression associated with karyotype in acute myeloid leukaemia |
Efficient uptake of fluorescently labeled miRCURY LNA ™ knockdown probe into Molt4 acute lymphoblastic leukaemia suspension cell line. Cells were electroporated (300V, 100 nM miRCURY LNA ™ knockdown probe, 5’ FAM labeled, 107 cells) and images were acquired 2 hours post-electroporation using a confocal microscope.
A : 5’ FAM labeled scrambled knockdown probe, FAM only
B : 5’ FAM labeled scrambled knockdown probe, FAM and DAPI
C : 5’ FAM labeled miR-specific knockdown probe, FAM only
D : 5’ FAM labeled miR-specific knockdown probe, FAM and DAPI
E : reagent only (no knockdown probe)