Download - Journal Club Presentation
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Nucleic Acids Research, 2007, Vol. 35, No. 8
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Intro:
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RNA Gel blot - discovery,validation & expression analysis of small regulatory RNA
straightforward and quantitative method
But less sensitive than cDNA cloning,RT-PCR or expression analysis by Microarray
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Problems:UV cross-linking - antagonistic to hybridization of small RNA
improved detection of small RNA cannot be achieved by increasing UV dosage
Instead, the UV step can be omitted
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UV Cross-linkingUV-cross-linking - thymine bases of DNA - the uridine residues
Uncharacterized reactive species - covalent cross-links with free amine of nylon membrane
exact mechanism is not known... :-(
water-soluble carbodiimide, 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide (EDC) can be used for efficient crosslinking
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Samples:Plants (siRNA)
Arabidopsis thaliana
Nicotiana benthamiana
Mammalian cells (miRNA)
HeLa cells
piRNA - piR1 from Mouse & Rat
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20 µg per lane - same RNA preparation from A. thaliana flowers
RNA hybridized at 50’C with a 32P–UTP-labelled RNA complementary to ath-mir-159b
membrane was washed in 0.1 SSC/0.1% SDS at 50’C
gamma-32P ATP-labelled Decade marker (M)
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The effect of varying UV dose on the detection of small
RNA.
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Improved detection of plant siRNA using EDC-mediated cross-linking
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RNA was extracted from leaves of transgenic N. benthamiana
- GFP 16C/GFPi transcribes a partial GFP inverted repeat (IR)
-from non-transformed N. benthamiana (NT).
Supplementary Data:
gel was stained - confirmed equal loading
- phosphorimaging of both the gel and the membrane
- demonstrate equal and efficient transfer of the gamma 32P-labelled marker RNA
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Figure shows:
(2–3-fold) marker RNA retained after cross-linking with EDC compared to UV.
approx. 30 fold increase in GFP siRNA detection
EDC cross-linking of siRNA was dependent on terminal phosphorylation
the signal eliminated by treatment of the RNA with alkaline phosphatase
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RNA was extracted from leaves of non-transformed N. benthamiana.
TS SINE retrotransposon siRNA
Half treated with alkaline phosphatase
0.5, 5 and 50 µg of both untreated (ut) and phophatased (ap) RNA run on the same gel
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EDC cross-linking improves detection of mammalian miRNA
cross-linked with 0.24 J UV or EDC at 40, 50 and 60’C for 15 min or 2 h.
(a) hsa-mir-21 /mmu-mir-292as
(b) has-mir-16 /mmu-mir-294s
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Improved detection of piRNA using EDC-mediated crosslinking
RNA extracted from
different organs.10µg loaded.
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Conclusions:EDC-cross-linking likely proceeds via a 5’ terminal phosphate
immobilized RNA that is much more amenable to detection by hybridization to probes
show that greatly improved detection of siRNA, miRNA and piRNA is possible with this modification
optimum, UV dosage for siRNA detection = optimum crosslinking
UV crosslinking = trade off between siRNA retention & detection
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Thank you13