Cat. No. / ID: 339115
Custom miRCURY LNA miRNA Detection Probes enable specific detection of novel miRNAs, miRNAs not covered by the predesigned miRCURY LNA miRNA Detection Probes, or other short, ncRNA sequences. These custom miRNA detection probes are LNA enhanced and are designed by our technical support scientists, according to the same design rules used for our predesigned miRNA detection probes.
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Custom miRCURY LNA miRNA Detection Probes are great for detecting any small RNA sequences, and they also serve as valuable controls in ISH experiments. Our LNA experts can design custom probes in a number of different ways so you can confirm signal specificity of your ISH experiment. As with any experiment, interpretation of the results is only as good as the controls. For ISH, there is no single best control, so it is preferable to have as many controls as possible to provide confidence in the observed expression pattern.
Mismatch probes are custom designed specifically for use alongside the target probe. A mismatch probe differs by 1–3 nucleotides towards the target. Our LNA design experts will identify the optimal number of mismatches and LNA positions, to reach a Tm value close to that of the target probe.
Using the same ISH protocol on serial sections, one should obtain little or no stain with the mismatch negative control probe. An example of a custom mismatch negative control probe can be seen in Figure 1F from the following publication: Nielsen et al. (2011). High levels of microRNA-21 in the stroma of colorectal cancers predict short disease-free survival in stage II colon cancer patients. Clin Exp Metastasis. 28(1):27–38. PMID: 21069438.
The short length of LNA probes enables the design of alternative probes that target slightly different regions, shifted by a few nucleotides, even within the short sequence of a mature miRNA (see figure Binding sites of four different LNA probes targeting the mature and precursor miRNA-21). Obtaining the same hybridization pattern with different probes targeting the same miRNA is a valuable positive control in ISH experiments. Our LNA experts can custom design alternative probes with LNA positions and a Tm value similar to that of the target probe.
Another valuable positive control probe to rule out non-specific cross hybridization is a loop-directed probe, designed to bind to the loop sequence of the precursor miRNA. This probe should confirm the same hybridization pattern as the target probe that binds to the mature miRNA sequence. However, the expression level of the pre-miRNA is likely to be lower than that of the mature miRNA.
An example of target-specific positive control probes, including a loop-directed probe, can be seen in Figure 1 from the following publication: Rask et al. (2011) High expression of miR-21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer. APMIS. 119(10):663-73. PMID: 21917003.
Binding sites of the miRCURY LNA microRNA Detection Probes correspond to the following probes: full length 22-mer miR-21 probe (gray), 17-mer probe (red), a 15-mer probe (blue), probe specific for the loop sequence (orange).