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