Matt:LabNotes/2014-5-16

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Detecting ppMALAT1 Hybridization[edit]

  • Test to see if dcProbe2RevComp-Cy3 (actually ATTO488) can accurately and efficiently detect hybridized padlock probes
    • Will try two methods, one where the padlock probe and dye-probe are added together and one where the dye-probe is added after padlock probe hybridization
    • Have negative control (dye-probe only) to detect for non-specific binding/false positive signal
  • Use sample from Matt:LabNotes/2014-5-7
    • A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2RevComp in one step
    • D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM dcProbe2RevComp after
    • D5: Negative Ctrl (1uM dcProbe2RevComp)

Procedure[edit]

  • Add 100ul of ppMALAT1 + dcProbe2-RevComp @45C for 24hrs

A3-A5

Component Volume
10X Ampligase Buffer 30
2uM ppMALAT1 1.5
100uM dcProbe2RevComp 3
H2O 265.5
Total 300

D3&D4

Component Volume
10X Ampligase Buffer 20
2uM ppMALAT1 1
H2O 179
Total 200

D5

Component Volume
10X Ampligase Buffer 10
100uM dcProbe2RevComp 1
H2O 89
Total 100
  • Aspirate D3&D4
  • Add 100ul 1uM dcProbe2RevComp in 30% formamide + 2X SSC for 10min @ RT
    • Preheated to 75C
  • Aspirate ALL
  • Wash with 2X SSC twice
  • Image (Saved in 5-17-2014)

Results[edit]

  • Counted rolonies with hybridized padlock probes with:
    • PISA7 (Gaussian std: 3, upper bound: -2e-4, area upper: 50, area lower: 4, axratio lower: .6, circ upper: 1.6, circ lower: .8, perim conn: 8, bkgmult: 7)
    • bwlabel (threshold of 9,000)
    • PISA7 Overlap (use PISA7 but only count ones that overlap with primary rolonies)

File:Hybridized PP Counts.JPG
Fraction of rolonies with hybridized padlock probes
File:Hybridized PP Ratio.JPG

Position 1st Rolonies PISA7 Rolonies PISA7 Overlap bwlabel Rolonies PISA7 Ratio Overlap Ratio bwlabel Ratio
1 930 779 468 261 0.838 0.503 0.281
2 1562 1250 782 433 0.800 0.501 0.277
3 3134 1446 1259 1018 0.461 0.402 0.325
4 4352 2067 1817 1427 0.475 0.418 0.328
5 4997 4458 3023 1633 0.892 0.605 0.327
6 9189 5975 4729 3695 0.650 0.515 0.402
7 1272 672 603 465 0.528 0.474 0.366
8 1062 591 520 354 0.556 0.490 0.333
9 3480 1766 1601 1229 0.507 0.460 0.353
10 3758 1689 1525 1414 0.449 0.406 0.376
11 3315 960 80 0 0.290 0.024 0.000
12 1909 427 17 2 0.224 0.009 0.001

Conclusion[edit]

  • PISA7 alone overcounts (false positive from noise) and bwlabel with threshold of 9,000 undercounts. PISA7 with only overlap seems most sensitive and accurate
  • ~50% of rolonies have hybridized padlock probes
    • Since only rolonies with >5 dyes can be detected, some rolonies probably have a couple padlock probes hybridized and not detected
    • Each of the detected rolonies have multiple padlock probes attached
  • There is non-specific binding as evidenced by the signal intensity from negative control