20100908 microRNA Presentation.pdf of biotechnology science

20100908 microRNA Presentation.pdf of biotechnology science

2. genome € y End

(533 microRNAs identified in humans) nsulinigtt sie miR-278 miR-14 a
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Approximately 2/3 of human genome is EA cali TD mace ne
regulated by microRNAs metabolism (postmitotic)
Longevity miR-143
Each microRNA regulates -200 target mnars
genes and multiple microRNAs can

regulate same gene

Patterns of microRNA expression define
tissue and cell type

miR-133. mi
microRNAs are responsible for stem cell PP on gies ¿/ IN”
differentiation N nn
AN y
F af Trighceride Cholesterol Myogenesis
microRNAs are critical in normal médiolen basynihess

development and etiology of disease Ay A2
Amino acid
Catabolism

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3. miRBase Sanger Institute, Cambridge, UK

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Dec 02

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sill

51 60 70 7.1 80 8182 9.09.1 9.2 Qo 19

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Currently- v. 10.4
Human = 541
Rat = 287
Mouse” = 443)

Virus (12)

66 different organisms
= Metazoa (35)

- Mycetozoa (1)

= Protista (1)

> Vitidiplantae (17)

Most extensive update
in microRNA sequences

HA À FRA

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4. Maturation
process

microRNA gene — primary transcript

primary transcript — “cropped” to stem-
loop microRNA precursor

microRNA precursor
cytoplasm
>

microRNA precursor — “diced” into
mature microRNA duplex

mature microRNA duplex — loaded into
RISC-like complex

gene expression down-regulated

5. How to detect miRNA?

7. RT primer

miRNA

PER ERR RRR )
cDNA

Step 2
Real-time PCR
\ Forward primer

TaqMan probe

Reverse
primer

ARA

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8. I Geiinto microRNA functional assay :
MR CIAN TIRNA Mimics and Inhibitors

Endogenous
Protein Expression

9. | Brsauer Details: miRIDIAN microRNA Mimic

10. Applications of miRIDIAN microRNA Mimics

11. ign

Optimal Desi

IMICS:

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12. RNA Mimic Positive Controls (1)

14. yA RS

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15. roauct Details: miRIDIAN microRNA Hairpin Inhibitors

16. Applications Of MIRIDIAN microRNA HP Inhibitors

17. From Vermeulen et al., RNA (2007) Double-stranded regions are essential design components of potent
inhibitors of RISC function

Flanking nucleotides
0 (RC)
2
4
6
8
10
12
14
16

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19. and. U's) for the flanking sequneces

Normalized Rluc/Fluc ratio

BY+RC+8Y

High
inhibition

3.125 nM
inhibitor €)

Low
inhibition

2

NAS Dd
eral different structures, and the hairpin gave the best inhibitio) KB A Ra
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20. High
inhibition

Normalized Rluc/Fluc ratio

3.425 nM
3.125 nM
3.125 nM
3.125 nM
3.125 nM

8Ahp+RC+8Ahp] 16A+RC+16A |IGAds+RC+16Ad RC Low
inhibition

Zn?

EA
We tried several different structures, and the halrpin gave the best Inhibition "I re ite RK

21. Matur
E miRIDIAN Hairpin
M CompettorE

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2nM 8AM 2nM 03nM|42nM 8n 5 7 ha 12

Mature AS Oligo. miRIDIAN Hairpin Days After Transfection
Inhibitor

+ microRNA Inhibitor function for miR-21 miRNA was assayed in
\ HeLa cells, 48 h after transfection using a dual-luciferase reporter
system, Each sample was normalized to matching treatment in

empty dual-luciferase reporte

FLD À IRD a

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22. N JE] _1sung using LIFE SCIENCE LID.

24. miRIDIAN M
loA mRNA expression va
miRNA. The miRIDIAN miRN,

mRNA levels even at 7 days, whe lard 2’-oMe ENA oligo complemintary to mature miR-122 showed a small effect. The

miRIDIAN miRNA Mimic immediately reduced Aldo A mRNA lelvels and sustained suppression throughout the experiment

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25. MIRIDIAN microRNA Summary

26. MAIDIAN microRNA Mimic and Hairpin Inhibitor Libraries

27. jt Phenotypic Screening: miRIDIAN

28. - MIRNA Library