Knockdown long, non-coding RNA genes with siRNA modified to ensure specificity
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Lincode siRNAs have been created to support the growing interest in analysis of long noncoding RNAs (lncRNA). As with siGENOME and ON-TARGETplus siRNAs, which target protein-coding genes, Lincode siRNAs are designed with the SMARTselection algorithm to ensure high-efficiency silencing. Lincode siRNAs also carry the proprietary ON-TARGETplus dual-strand chemical modifications to ensure optimal strand loading and disrupt microRNA-like seed activity for the reduction of off-targets.
Long noncoding RNA (lncRNA) has recently gained attention as a new species of regulatory RNA; with key roles in epigenetics, transcriptional regulation, development, cancer, neurological disorders, and other essential biological processes. Effective tools to silence lncRNAs will more fully elucidate the role of these molecules in genetic pathways.
The Lincode siRNA product line targets human and mouse noncoding RNA genes in the RefSeq database that meet the following criteria:
- ≥200 nt in length
- Gene record contains at least one RefSeq Accession prefix of NR_ or XR_
- RNA type "noncoding RNA" or "miscellaneous RNA" to exclude known classes like tRNA, rRNA, etc. microRNA, another type of non-coding RNA, is modulated by reagents available in the miRIDIAN product line)
What about non-coding transcripts within protein-coding gene records?
A large number of protein-coding genes contain at least one non-coding (NR) transcript in addition to its protein-coding transcripts (NM or XM). Lincode siRNAs have been developed to specifically target these non-coding transcripts when unique sequence space is available.
lncRNA may be more difficult to silence - or its knockdown may be more difficult to detect than protein-coding genes due to:
- Nuclear localization of the target, making it less available to RNAi cellular machinery
- Secondary structure of the lncRNA, preventing siRNA access to the target region
- DNA or protein binding, preventing siRNA access to the target region
What about knockdown of a lncRNA that isn't in RefSeq?
To design and order siRNAs targeting lncRNA that fall outside of the Lincode predesigned products, follow these steps:
- Go to the siDESIGN Center and enter the RefSeq ID or nucleotide sequence of the lncRNA you wish to target
- Select the BLAST database that includes NM and NR (coding and non-coding transcripts) for your species of interest
- Add desired designs to your cart, then click each item to select ON-TARGETplus modifications to ensure strand-specific silencing
Lincode control siRNA reagents incorporate specificity-enhancing modifications to improve experimental results. Non-targeting siRNAs are designed with three or more mismatches to every lncRNA and protein-coding transcript in human, mouse, and rat. GAS5 is a widely expressed lncRNA whose knockdown has been demonstrated in multiple cell lines. If GAS5 expression levels are undetermined in your cell type, the use of ON-TARGETplus positive controls is an alternative method for initial experimental optimization.
Lincode positive control siRNA
A positive control siRNA targeting the GAS5 long noncoding RNA (lncRNA) in human cells. Includes patented dual-strand modifications to minimize off-target effects. Useful for determination of optimal RNAi conditions.
A positive control pool of four siRNAs targeting the GAS5 long noncoding RNA (lncRNA) in human cells. Includes patented dual-strand modifications to minimize off-target effects. Useful for determination of optimal RNAi conditions.
Lincode negative control siRNA
Negative control siRNAs with three or more mismatches to any human, mouse or rat lncRNA or protein-coding gene. Dual-strand modifications reduce potential off-targets. Ideal for determination of baseline cellular responses in RNAi experiments.
Negative control pool of four siRNAs with three or more mismatches to any human, mouse or rat lncRNA or protein-coding gene. Dual-strand modifications reduce potential off-targets. Ideal for determination of baseline cellular responses in RNAi experiments.
No predesigned product to fit your needs? Use our online design tools and extensive synthesis options to create a custom siRNA specific for your application.
Unbiased RNAi screens have become an important tool for elucidation of novel biological pathways and disease progression. The assessment of potential roles for microRNA and lncRNA, in addition to protein-coding genes, can provide a more complete picture of the genes involved in the phenotype of interest.
Human and Mouse Lincode siRNA screening libraries are available as SMARTpool or a Set of 4 individual siRNAs to support efforts to decipher lncRNA function.
3430 total long noncoding RNA gene targets containing at least one NR accession number, includes siRNAs targeting NR transcripts within protein coding genes.
1997 total long noncoding RNA gene targets containing at least one NR accession number, includes siRNAs targeting NR transcripts within protein coding genes.
Lincode siRNA reagents available as plated libraries for any customer-defined collection of lncRNAs of interest.
- A mixture of 4 siRNA provided as a single reagent; providing advantages in both potency and specificity.
Set of 4:
- Discounted price and one-click purchase of the four siRNAs targeting the same gene. No prior purchase required.
- Select 1, 2, or 3 individual siRNAs per gene. Minimum purchase of four siRNAs (any gene, any product line) required at the 2 nmol size.
Lincode siRNAs effectively knockdown target lncRNAs
Detection of effective lncRNA knockdown following application of Lincode siRNA reagents. All siRNAs used at 25 nM. Detection of remaining lncRNA by corresponding Solaris (sold under the Thermo Scientific brand) qPCR lncRNA expression assay.
lncRNAs demonstrate highly variable expression/uploadedImages/Products/RNA_Interference/sirna/lincode/_assets/lincode.PNG
Robust qPCR assays are necessary for accurate determination of lncRNA expression levels. It is of vital importance to determine lncRNA expression in your cells prior to knockdown experiments. lncRNA expression can be expected to vary much more than is typically observed from protein-coding genes like Cyclophilin B (PPIB).
lncRNAs demonstrate lower and more variable expression than protein-coding genes. It is therefore highly recommend to determine lncRNA expression levels in your cells prior to knockdown experiments. Solaris qPCR expression assays are designed for sensitive detection of lncRNA and demonstrate high sensitivity and reproducibility.
50 ng of total RNA was converted into cDNA using the Maxima First Strand cDNA Synthesis Kit (#K1641). Gene expression levels were evaluated with Solaris qPCR assays. Cyclophilin B (PPIB) was used as a reference for typical mRNA expression. Standard RT-PCR cycling conditions resulted in initial Cq values ranging from 22 (PPIB in 293T cells) to 36 (BDNF-AS1 in HeLa cells). MLK7-AS1 lncRNA expression was not detectable in any of the cell lines tested. Cell lines include HeLa, HEK293T, Human Neonatal Dermal Fibroblast (hNDF).
Modifications to enhance function and reduce off-targets
Lincode siRNAs are modified with a proprietary dual-strand modification that improves siRNA specificity.
Additionally, off-targets are reduced due to:
- Inactivation of sense strand activity; driving preferential loading of the antisense strand into RISC
- Novel antisense seed region modification for disruption of microRNA-like off-targets
Sense strand activity is prevented by siRNA modifications
BDNF-AS1 lncRNA is anti-sense to BDNF protein-coding RNA. Direction of transcription from genomic DNA is indicated by arrows, exons are indicated by rectangles. Position of the Lincode siRNA target is indicated by double green lines.
Lincode siRNA modifications prevent sense strand activity
qPCR results indicate of a protein coding transcript (BDNF) with Lincode siRNA targeting a lncRNA on its antisense strand (BDNF-AS1), indicating strand specificity of lncRNA siRNA design and effectiveness of ON-TARGETplus modifications.
Lincode siRNA modifications greatly reduce Off-targets
To detect subtle phenotypic changes that may arise from lncRNA knockdown, it is essential to incorporate strategies to prevent the off-targeting of protein-coding genes. Lincode siRNA reagents are synthesized with proprietary dual-strand modifications known as the ON-TARGETplus modification pattern which has been proven to reduce off-targets arising from microRNA-like activity of the antisense seed region of the siRNA.
Two different siRNAs targeting the same lncRNA were synthesized with modifications that (a) block the sense strand only or (b) the ON-TARGETplus modification pattern which blocks the sense strand and includes an antisense strand seed region modification. While the target lncRNA was effectively silenced by all four siRNAs (arrows), the siRNAs synthesized with the ON-TARGETplus modifications demonstrated greatly reduced off-targets.
Agilent™ G3 Human Gene Expression Microarray™ HeLa 12K, harvest 24 hrs post transfection, 100 nM siRNA, Analysis: 2 fold down regulated, pval 0.05, and non-siRNA-specific effects filtered out.