Nucleic Acids Research, 2007, Vol. 35, No. 8
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Intro:
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-linking
UV-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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The effect of varying UV dose on
20 µg per lane - same RNA
the detection of small
preparation from A. thaliana
RNA.
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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Improved detection of plant siRNA
using EDC-mediated cross-linking
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 you
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