Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.

1. Kim Lennox, MSc
Research Scientist, Integrated DNATechnologies
Methods to knock down lncRNAs
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2. • Lower organisms: mRNA > lncRNA
• Higher organisms: lncRNA >> mRNA
• Added regulatory functions of
lncRNAs:
– Correlate with an increased ability
of multicellular organisms to
differentiate into many different
cells types
– Allow an organism to achieve
greater diversity from the same
number of protein coding genes
Importance of lncRNAs
in mammals
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3. Target Location
mRNA cytoplasm + nucleus
splice nucleus only
miRNA cytoplasm > nucleus
lncRNA
cytoplasm > nucleus
cytoplasm = nucleus
cytoplasm < nucleus
Does cellular localization matter when
choosing a silencing reagent?
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4. Rinn study: RNA-FISH on 61 lncRNAs
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Cabili et al. (2015) Localization and abundance of human lncRNAs at single-cell and single molecule
resolution. Genome Biol, 16:20.
Cell lines: HeLa, hlF, hFF

5. 5
RISC
Target RNA
RNA cleavage
siRNA
RNA cleavage
Target RNA
RNA interference (RNAi)
Target RNA
RNase H1
ASO (DNA)
Antisense oligonucleotides (ASOs)
> cytoplasm > nucleus

6. Study design
• lncRNA targets
– 2 nuclear
– 3 cytoplasmic
– 3 nuclear + cytoplasmic
• Knockdown reagents
– Antisense oligonucleotides (ASOs)
• 12 DNA-PS (20mers)
• 12 2′OMe-PS 5-10-5 chimeras (same sequence as DNA-PS)
• 6 LNA™ longRNA GapmeRs (sites selected by Exiqon)
– siRNA
• 12 Dicer-substrate siRNAs (DsiRNAs) (27mer, IDT design)
• 12 siRNAs (21mer,Thermo Fisher design)
• 4 Silencer® Select siRNAs (21mer, LifeTechnologies design)
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7. The knockdown reagents
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8. Methods
• Transfection (ASOs and siRNAs):
– HeLa cells (Huh7 for NRON); all data replicated in HCT116 cells
– Biological triplicates
– 96-well format
– Lipofectamine® 2000
– Chemistry-matched negative control sequences
• RNA was prepared 24 hours post-transfection
• RT-qPCR:
– Two qPCR assays for each target (one towards the 5′ end and one towards the 3′ end)
– Triplicate qPCRs
– Quantification standard curves on each 384-well plate
– Results normalized against internal control HPRT and SFRS9 gene expression levels
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9. Targets
Nuclear Cytoplasmic Nuclear + Cytoplasmic
Target 1: MALAT1 (8 kb) Target 3: NRON (2.7 kb) Target 6: TUG1 (7.5 kb)
Target 2: NEAT1 (3.7 kb) Target 4: DANCR (0.9 kb) Target 7: CasC7 (9.3 kb)
Target 5: OIP5-AS1 (1.9 kb) Target 8: HOTAIR (2.3 kb)
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10. In situ hybridization: MALAT1 (nuclear)
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11. In situ hybridization: NEAT1 (nuclear)
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12. In situ hybridization: CasC7 (nuclear +
cytoplasmic)
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13. MALAT1 knockdown in HeLa cells: nuclear
For mRNA knockdown, we usually expect >80% success rate for the DsiRNA algorithm.
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14. NEAT1 knockdown in HeLa cells: nuclear
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15. NRON knockdown in Huh-7 cells: cytoplasmic
NRON function involves tight binding of multiple protein species → no knockdown.
Therefore, needed to move to a different cytoplasmic target. 15

16. DANCR knockdown in HeLa cells: cytoplasmic
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17. OIP5-AS1 knockdown in HeLa cells: cytoplasmic
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18. TUG1 knockdown in HeLa cells: nuclear + cytoplasmic
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19. CasC7 knockdown in HeLa cells: nuclear + cytoplasmic
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20. HOTAIR knockdown in HeLa cells: nuclear + cytoplasmic
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21. Comparison summary
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2′OMe-PS ASO
LNA-PS ASO
LNA-siRNA
DsiRNA
siRNA

22. Combinatorial approach can have additive effects for
lncRNAs localized in both the nucleus and cytoplasm
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0
20
40
60
80
100
120
ASO DsiRNA ASO +
DsiRNA
ASO DsiRNA ASO +
DsiRNA
ASO DsiRNA ASO +
DsiRNA
%RemainingRNA
Combinatorial Knockdown
10 nM
5 nM
2.5 nM
1 nM
MALAT1
Nuclear
OIP5-AS1
Cytoplasmic
CasC7
Both

23. Comparison of ASO chemistry/design potency at the same 6
sites in MALAT1
2nd generation “Gapmer”ASOs are more potent than DNA-PS
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24. Summary
• Overall performance of ASOs vs. siRNAs varies with the dominant cellular
localization of the lncRNAs that were targeted:
– ASOs were more often effective at knocking down nuclear lncRNAs.
– siRNAs were more often effective at knocking down cytoplasmic lncRNAs.
– Better knockdown can be achieved by combining RNAi reagents with ASOs.
• Characterizing the localization of a targeted lncRNA and selecting the
appropriate knockdown method is important for improving the success of the
knockdown experiment.
• If lncRNA localization is unknown, trying both methods may be prudent.
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25. Articles from IDT scientists:
• www.idtdna.com/decoded (Small RNAs/FunctionalGenomics section)
– A New Renaissance for Antisense in the Era of lncRNA
– Using AntisenseTechnologies to Modulate Noncoding RNA Function
• www.idtdna.com (Search for “antisense oligonucleotide”)
– Antisense Oligonucleotides: Strategies and Applications
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26. Online tools:
• www.idtdna.com/scitools (Gene Regulation and Knockdown section)
– Predesigned DsiRNA SelectionTool
Selects DsiRNA Duplexes andTriFECTa® Screening Kits for your sequence
– RNAi DesignTool
Generates duplex siRNA sequences for RNAi applications
• For additionalASO design assistance, please email
ApplicationSupport@idtdna.com
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