Matt:LabNotes/2014-2-25

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Secondary Rolony Generation[edit]

Samples: Matt:LabNotes/2014-2-21

Procedure[edit]

Anneal Padlock Probes[edit]

  • 1X Ampligase Buffer
  • 2uM of each padlock probe
  • 0.5 U/ul Ampligase
  • Incubate @ 45C for 24 hours
    • Wrapped in parafilm and put in ziploc with wet towel
Fixed Template - 25U Ampligase (C9 & F9)[edit]
Component Volume
10X Ampligase Buffer 10
200uM ppMALAT1 1
5U/ul Ampligase 10
H2O 79
Total 100
Fixed Template - 50U Ampligase (C10 & F10)[edit]
Component Volume
10X Ampligase Buffer 10
200uM ppMALAT1 1
5U/ul Ampligase 20
H2O 69
Total 100
1st Rolony (E6 & F6)[edit]
Component Volume
10X Ampligase Buffer 10
200uM ppMALAT1 1
200uM ppACTB 1
200uM ppRAB7A 1
5U/ul Ampligase 10
H2O 77
Total 100
Empty Negative Control (D7 & E7)[edit]
  • Not going to waste Ampligase since it's highly unlikely anything will be ligated, but will do RCA in these wells
Pre-circularized Padlock Probes (C7 & C8)[edit]
  • Combined all the pre-circle ppMALAT1 made on: Matt:LabNotes/2014-2-10 into 64ul
  • 75% EtOH for 10 minutes
  • Wash with 1X PBS 2 times
  • 0.1N HCl for 2 minutes
  • Wash with 1X PBS 3 times
  • Add 32ul to C7 & C8
Fixed Pre-circularized Padlock Probes (F7 & F8)[edit]
  • Not going to do anything during Ampligase step

Secondary RCA[edit]

  1. Aspirate each well but don't wash
    • F7 well was dried up
  2. Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
    1. 3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of 2xSSC/30% formamide
    2. Preheat primer at 60C (Tm = 55C when not in 30% formamide)
    3. Add 50ul to each well, incubate at 45C for 15min.
    4. Aspirate, and wash using 2x SSC twice
    5. Aspirate, and wash using 0.1x SSC twice
  3. Prepare the rolling circle amplification reaction mixtures on ice.
    1. 513 ul ddH2O,
    2. 60 ul 10x Phi29 buffer,
    3. 6 ul 25 mM dNTP,
    4. 12 ul 2 mM aminoallyl dUTP
    5. 9 ul Phi29 DNA polymerase (Epicentre, low concentration)
      • 600ul Total Volume
  4. Add 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14)
  5. Wash 1X PBS once
  6. Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS)
  7. Wash 1X PBS twice
  8. Add 50ul 1M Tris pH 8.0 @RT for 1hr
  9. Wash 1X PBS twice

Detection[edit]

  • To all wells add 50ul 1uM dcProbe2-Cy3 (6ul 100uM dcProbe2-Cy3 + 180ul 100% formamide + 60ul 20X SSC + 354ul H2O preheated to 75C)
    • Strip E6 & F6 with 80% formamide @ 37C
  • To E6 & F6 add 50ul 1uM dcProbe0-Cy3 (10ul 10uM dcProbe0-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
    • Strip with 80% formamide @37C
  • To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
  • Images saved under 2-27-2014 folder

Results[edit]

Positive Control - Fixed Template (F9 & F10)[edit]

  • Comparing # of Fixed Template with # of 2nd Rolonies Generated
    • F9 used 0.5U/ul Ampligase while F10 used 1U/ul
    • When using MALAT1_1stRolonyFISH to count Fixed Templates, only counted large clumps of targets while single targets may not be bright enough to see
' # Fixed Template # 2nd Rolonies
F9 (0.5 U/ul) 30 5,624
F10 (1 U/ul) 61 7,883
  • Secondary Rolonies were many! Looks Great!
    • Here is F9 example PISA7 counted (red marks counted rolonies) image while raw tif can be found in powerpoint

File:F9 dcProbe2 G550 Cy3 ch00 MIP PISA7Overlay.png

Ampligase Concentration Comparison: 0.5U/ul vs 1U/ul[edit]

  • Looking at # of 2nd Rolonies Generated and assuming about same # of fixed template in each
    • C9 & F9 are 0.5U/ul
    • C10 & F10 are 1U/ul
  • No significant difference so will keep using 0.5U/ul
Ampligase Conc 0.5 U/ul 1 U/ul
Row C 5,532 4,536
Row F 5,624 7,883

Positive Control - Fixed Precircularized ppMALAT1_NH2 (F7 & F8)[edit]

  • It was hard to tell if any was fixed in the first place with dcProbe2RevComp-Cy3
  • 2nd Rolony results seem to confirm that since there was less 2nd Rolonies than even the negative control
' # Fixed Circle # 2nd Rolonies
F7 9 (high gain) 5
F8 12 (high gain) 11

Positive Control - Not Fixed Precircularized ppMALAT1 (C7 & C8)[edit]

  • C8 had ~5-10X more secondary rolonies than C7 but both were way more than what we've seen before
    • No point actually counting since there's nothing to compare to
  • Example of C8

File:C8 dcProbe2 G550 Cy3 ch00 MIP.png

Negative Control (D7 & E7)[edit]

  • Saw more signal than many 2nd Rolony Ampligase tests from before so should do negative control every time in the future to get a baseline of false positives
  • D7: 43
  • E7: 28

Experimental Sample (E6 & F6)[edit]

' E6 Targets E6 2nd Rolonies F6 Targets F6 2nd Rolonies
ACTB 6 28 6 13
RAB7A 7 46 6 15
MALAT1 5 246 4 145
  • Based on the MALAT1 results it seems that trying to quantify targets with 1stRolonyFISH probes isn't very accurate
  • Since the 2nd Rolonies of ACTB and RAB7A are in the range of Negative control we can't confidently say any ACTB or RAB7A padlock probes produced 2nd Rolonies
  • It's curious that only MALAT1 seemed successful
    • Could be that dcProbe2-Cy3 (recently ordered from IDT to replace the old dye from Illumina) which was used to detect MALAT1 works better
    • Could also be MALAT1 is more abundant than the other 2, which is what was reported in Harvard's FISSEQ Science paper