Our best recommendations for silencing your mRNA or lncRNA of interest using Antisense LNA™ GapmeRs, and successfully validating the knockdown using ExiLERATE LNA™ qPCR.
1. Know your target
A single gene often gives rise to multiple transcripts. Multiple copies of genes and overlapping sense/antisense transcripts add to the complexity of gene expression.
Exiqon’s online design tools help you to choose Antisense LNA™ GapmeRs and ExiLERATE LNA™ qPCR assays that target exactly your transcript(s) of interest – and avoid misleading results from related isoforms or transcript variants.
Antisense LNA™ GapmeRs are single stranded oligonucleotides, therefore strand-specific knockdown is guaranteed. Exiqon’s LNA™ GapmeR design algorithm is optimized to identify the most specific LNA™ GapmeRs by incorporating alignments to both the spliced and unspliced transcriptomes.
ExiLERATE LNA™ qPCR assays are Tm normalized, enabling optimal primers to be designed to precisely amplify any region of a transcript. This is useful to detect specific isoforms or transcript variants. In addition, ExiLERATE LNA™ qPCR assays are optimized to avoid amplifying the opposite strand.
LNA™ tools for knockdown and validation of RNA silencing
Figure 1. Antisense LNA™ GapmeRs and ExiLERATE LNA™ qPCR assays can be designed to target exactly your transcript(s) of interest. This avoids misleading results from related isoforms or transcript variants.
2. Validate knockdown with more than one qPCR assay
This is especially recommended for poorly annotated transcripts, genes with multiple transcripts or when knocking down longer transcripts.
3. Normalize to multiple reference genes
Robust normalization is key for accurate measurement of target RNA knockdown. Test several candidate reference genes using validated ExiLERATE LNA™ qPCR Control primer sets, and use GeNorm or NormFinder in Exiqon’s GenEx Software to identify the most stable combination for robust normalization.
4. Use validated positive controls
Exiqon’s validated positive control Antisense LNA™ GapmeRs and corresponding validated ExiLERATE LNA™ qPCR Control primer sets help to optimize delivery conditions.
5. Use a negative control
One of Exiqon’s Negative control Antisense LNA™ GapmeRs can be used to confirm the phenotype is due to target RNA knockdown. An untreated control is also useful to evaluate effects of transfection.
Concerning ExiLERATE LNA™ PCR: NOTICE TO PURCHASER: LIMITED LICENSE Purchase of the products includes an end-user license for research use only under patents specified in the product insert. No other patent rights are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. For life science research use only. Not for use in diagnostic procedures.