Lincode siRNAs  

Lincode siRNAs

Product Overview

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Lincode siRNAs

 

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.

 

Product Formats

SMARTpool:

  • 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.

Individual siRNAs:

  • 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.
 
 

Supporting Data


Knockdown of CDKN2B-AS1 in HeLa cells

Effective Knockdown

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.

Knockdown of CDKN2B-AS1 in HeLa cells

(A) Knockdown of CDKN2B-AS1 in HeLa cells; normalized to Lincode Non-targeting siRNA #4 (D-001320-04)

Knockdown of BDNF-AS1 in hNDF cells

(B) Knockdown of BDNF-AS1 in hNDF cells; normalized to Lincode Non-targeting siRNA #2 (D-001320-02)

Expression analysis of nine lncRNAs varies widely across three human cell lines

Variable Expression

lncRNAs demonstrate highly variable expression

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).

Expression analysis of nine lncRNAs

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

Enhanced Specificity

Modifications to enhance function and reduce off-targets

Lincode siRNAs are modified with a proprietary dual-strand modification that improves siRNA specificity

Modifications to enhance function and reduce off-targets

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
 
Lincode siRNA modifications prevent sense strand activity

Importance Of Strand Specificity

Sense strand activity is prevented by siRNA modifications

Genomic context and Lincode siRNA targeting of BDNF-AS1 lncRNA.

Genomic context and Lincode siRNA targeting of BDNF-AS1 lncRNA

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.

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.

Modifications to enhance function and reduce off-targets

Reduced off-targets

Lincode siRNA modifications greatly reduce Off-targets

Genomewide expression analysis demonstrates improved specificity of Lincode siRNA.

Modifications to enhance function and 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.

 
 
 

Experimental Considerations

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 pre-designed 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

Still have questions? Need more assistance? Check out the siDESIGN User Guide or contact us.