Detecting small rnas

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Detecting small rnas
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Techniques

• Sequence homology to closely related bacteria in
  intergenic regions (4 Salmonella, K pneumoniae, Y
  pestis)
• BLASTed all Ig regions gt 180bp and gave score
  from 0-5 based on BLAST score and length
• Used information on location of conserved region
  w/ respect to bordering genes (conserved regions
  next to 5 end probably regulatory region)

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Sequence homology
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Techniques

• Microarray expression data using Affymetrix chip
• Bonus points for detecting expression in Ig
  region on opposite strand of bordering coding
  regions
• Added rifampicin 20 minute prior to harvesting
  rna to look for stable rnas

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Microarray Expression
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All canidates
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Techniques

• Performed Northern blot to find srna
• Created labeled complimentary probes for 61
  candidates
• Ran all rna on gel, used probe to identify if
  candidate was present and how long
• Used length and 3rd base pair conservation to
  determine if orf
• Used three conditions, early exponential, mid
  exponential, and stationary

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Northern blot
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Interaction w/ Hfq protein

• Performed Immunoprecipitation experiment w/ Hfq
• Crosslink everything together
• Grab all the Hfq proteins using Hfq antibody
  attached to beads
• Reverse crosslink
• Perform northern on remaining rna

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Northern blot
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Results

• 15 novel srnas identified (of 60)
• 2 predicted srnas that werent seen in any
  northern
• 6 new short orfs
• 4 of 15 srnas showed no microarray detection

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Problems

• Need closely related species to your species of
  interest to be sequenced
• Affymetrix chips are expensive
• Need to test many conditions to make the srna
  expressed. Can only give positive result
• Rifampicin didnt seem to work to well (2 out of
  15)
• Ig regions must be gt 180 nt