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Nucleic Acids Research, 2007, Vol. 35, No. 8

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

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.

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

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