Journal Club Presentation


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A presentation that i gave during my Journal Club.Its always good to keep the presentation short and to the point.

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Journal Club Presentation

  1. 1. Nucleic Acids Research, 2007, Vol. 35, No. 8 1
  2. 2. 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 2
  3. 3. 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 3
  4. 4. 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 4
  5. 5. Samples: Plants (siRNA) Arabidopsis thaliana Nicotiana benthamiana Mammalian cells (miRNA) HeLa cells piRNA - piR1 from Mouse & Rat 5
  6. 6. 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) 6
  7. 7. 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 7
  8. 8. 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 8
  9. 9. 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 9
  10. 10. 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 10
  11. 11. Improved detection of piRNA using EDC-mediated crosslinking RNA extracted from different organs.10µg loaded. 11
  12. 12. 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 12
  13. 13. Thank you 13