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5-2014 RNAi Workshop-lncRNA Tools
5-2014 RNAi Workshop-lncRNA Tools
1. Proteins coding: 2%
Detection of lncRNAs
• Design of qPCR assays
3
Outline
Background
• What are lncRNAs?
Detection of lncRNAs
• Design of qPCR assays
4
Non-coding RNA (ncRNA)
mRNA
Non-coding RNA
Protein
5
Protein-coding genes vs. Bio-complexity
~ 21,000
Human C. elegans
3%
% of genome
encoding proteins
non-coding
Human C. elegans
3%
% of genome
encoding proteins
non-coding
1. Housekeeping RNA
tRNA, rRNA, snoRNA, snRNA
Translation, ribosome mature, RNA splicing…etc
(1) De Sacco et. al. Int. Journal of Mol. Sci. (2012), 13.
(2) Taft et al., J Pathol 2010; 220: 126–139, Non-coding RNAs: regulators of disease
10
Genomic locations of lncRNAs
Transcriptional interference
X-chromosome inactivation
XIST compensates dosage by silencing an X-
chromosome in females
Regulating imprinting
Air recruits histone methyl-transferase to silence
genes in the Igf2r locus during early development
Epigenetic regulation
Functional demarcation of active and silent
chromatin domains in human HOX loci by
noncoding RNAs [HOTAIR]
Rinn et al., Cell 2007
17
lncRNAs in human disease
HIV Replication
NEAT1 Long Noncoding RNA and Paraspeckle
Bodies Modulate HIV-1 Posttranscriptional
Expression
mBio 2013
Alzheimer’s Disease
Knockdown of BACE1-AS Nonprotein-coding
Transcript Modulates Beta-Amyloid-Related
Hippocampal Neurogenesis
Int J Alzheimers Dis. 2011
Melanoma
Melanoma-upregulated SPRY4-IT1 Modulates
Apoptosis and Cell Invasion
Cancer Research 2011
18
The rise in the number of lncRNA publications
2013
2013
21
Example – Role of NEAT1 lncRNA in HIV infection
23
Outline
Background
• What are lncRNAs?
Detection of lncRNAs
• Design of qPCR assays
24
Sources for design of qPCR assays
25
Considerations for design of qPCR assays
Splice Variant 2
Splice Variant 3
26
Ideal qPCR lncRNA expression assays
• Probe and primer sets increase specificity
27
Examination of lncRNA Expression
High Cq =
Low expression
Low Cq =
High expression
28
Outline
Background
• What are lncRNAs?
Detection of lncRNAs
• Design of qPCR assays
29
Non-coding RNA (ncRNA)
5’-p RISC
p-5’
mRNA
Non-coding RNA
RISC p-5’
Protein
30
Challenges for lncRNA knockdown
31
Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown
32
Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown
33
siRNA design algorithm
34
Ex. siRNA-mediated Knockdown of 15 lncRNAs
35
Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown
36
Use of siRNA pool
37
Use of siRNA pool
38
Effective knockdown of lncRNAs
39
Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown
40
Chemical modification select RISC-loading strand
S AS siRNA
S BDNF RNA
AS BDNF-AS1 RNA
RISC
AS
S BDNF RNA
RISC or
S
AS BDNF-AS1 RNA
41
Dual-strand modifications to improve specificity
42
Chemical modifications reduce off-targets
S AS siRNA
S BDNF RNA
AS BDNF-AS1 RNA
RISC
AS
S BDNF RNA
RISC or
S
AS BDNF-AS1 RNA
43
Chemical modifications reduce off-targets
S AS siRNA
S BDNF RNA
AS BDNF-AS1 RNA
RISC
AS
S BDNF RNA
RISC or
S
AS BDNF-AS1 RNA
44
Chemical modifications reduce off-targets
RISC
S
AS BDNF-AS1 RNA
45
Strategies to improve Function and Specificity of
siRNAs for lncRNA knockdown
46
Discussion and Questions
47
FAQs
http://www.thermoscientificbio.com/design-center/
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Q & A siRNA siDESIGN Center
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Q & A siRNA siDESIGN Center
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Q & A siRNA siDESIGN Center
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