Matt:LabNotes/2016-8-21

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SplintR Ligase Test

Reference

Experimental Outline

  1. Remove DNA from RNA (UHRR)
    • Shouldn't be completely necessary
  2. Biotinylate RNA
  3. Attach RNA to magnetic streptavidin beads
  4. V6 Padlock Probe capture to RNA
  5. Quantify captured products by qPCR
    • Also adds Illumina sequencing adapters

Sample Groups

  • Use Universal Human Reference RNA (UHRR)
  • 3 RNA template concentrations for each (30ng, 150ng and 820ng)
    • For DNA template positive control only do 1 sample using 300ng gDNA
  • Sample 1: gDNA template captured by V6 padlock probes with Ampligase at 95C->55C
    • Standard padlock probe capture
  • Sample 2: RNA template captured by V6 padlock probes with SplintR at 37C
    • Should be ligated but non-specific because low temp
  • Sample 3: biotinylated and streptavidin bead linked RNA captured by V6 padlock probes with SplintR at 37C
    • No washing or pull down
    • Should be same as Sample 2 unless RNA is lost during biotin or streptavidin bead steps
  • Sample 4: RNA template captured by V6 with Ampligase at 37C
    • Negative control to see difference between SplintR and Ampligase
  • Sample 5: No template with V6 and SplintR at 37C
    • NTC checks for ssDNA ligase activity
  • Sample 6: biotinylated and strept bead linked RNA captured by V4 padlock probes at 55C followed by wash and then SplintR & gDNA at 37C
    • V4 padlock probes should be targeting wrong strand and completely washed away before
  • Sample 7: biotinylated and strept bead linked RNA captured by V6 at 55C followed by wash and then SplintR at 37C
  • Summary:
    • 3 samples with biotinylated RNA
    • 2 samples where RNA is pulled down by magnetic beads and washed to remove non-specific binding

Experiment

Remove DNA from RNA

  • Skipped this step

Biotinylate RNA

  • Use Pierce RNA 3' End Biotinylation Kit (>75% ligation efficiency)
  • 3ul of 1ug/ul UHRR
  1. Thaw all kit components except 30% PEG and DMSO on ice
    • Thaw DMSO at RT and 30% PEG at 37C for 5-10min
  2. Adjust heating block to 85C
  3. Transfer 3ul RNA to 1.5ml tube and heat for 3-5min at 85C
  4. Place RNA immediately on ice
  5. Prepare labeling reaction mix by adding in order listed
    • Use new pipette tip to mix reaction mix after PEG addition
Component Volume
nf H2O 5
10X RNA Ligase Buffer 3
Rnase Inhibitor 1
RNA 3
Biotinylated Cytidine (Bis)phosphate 1
T4 RNA Ligase 2
30% PEG 15
Total 30
  1. Incubate at 16C for overnight (~15 hrs)
  2. Add 70ul of nf-H2O
  3. Add 100ul chloroform:isoamyl alcohol to extract RNA ligase
    • 96ul chloroform + 4ul isoamyl alcohol
    • Vortex briefly and then centrifuge 2-3 min at high speed to separate phases
  4. Carefully remove the top (aqueous) phase and transfer to a new tube
  5. Add 10ul 5M NaCl, 1ul glycogen, and 300ul ice-cold 100% EtOH
  6. Precipitate for 1hr at -20C
  7. Centrifuge for 15min at > 13,000 x g at 4C
  8. Remove supernatant
  9. Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH
  10. Air-dry for ~5min
  11. Resuspend pellet in 20ul nf-H2O (or 2X B&W buffer for binding to Streptavidin beads)

Attach RNA to Streptavidin Beads

  • MyOne C1 protocol
  • Make Solutions A & B and Binding&Wash Buffer
    • Solution A (DEPC-treated 0.1 M NaOH, DEPC-treated 0.05 M NaCl): 2.5ml 1M NaOH + 0.25ml 5M NaCl + 22.25ml nf-H2O
      • NaOH: 39.997g/mol -> 1M NaOH = 0.1g NaOH dissolved in 2.5mL nf H2O
    • Solution B (DEPC-treated 0.1 M NaCl): 0.5ml 5M NaCl + 24.5ml nf-H2O
    • 2X B&W Buffer (10 mM Tris-HCl 1 mM EDTA 2M NaCl): 10ml 5M NaCl + 50ul 0.5M EDTA + 62.5ul 4M Tris-HCl + 14.8875ml H2O
  1. Resuspend beads by vortexing
  2. Transfer 100ul of beads (10ug/ul) to new tube
  3. Pull down by magnet 2min and remove supernatant
  4. Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant
  5. Wash 2x with 100ul Solution A for 2 min each
  6. Wash 2x with 100ul Solution B
  7. Resuspend the beads in Solution B
  8. Add biotinylated RNA
  9. Incubate 15min at RT gently rotating
  10. Pull down with magnet for 3min
  11. Wash 3x with 1X B&W Buffer
  12. Resuspend in 1ml 1X PBS

V6 Padlock Probe Capture

  • SplintR at 37C or 25C? 4hrs or overnight?

qPCR