Matt:LabNotes/2014-5-16: Difference between revisions
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>Mzcai (Created page with "==Detecting ppMALAT1 Hybridization== *Use sample from Matt:LabNotes/2014-5-7 **A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2-RevComp in one step **D3 & D4: Hybridi...") |
>Mzcai m (→Procedure) |
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==Detecting ppMALAT1 Hybridization== | ==Detecting ppMALAT1 Hybridization== | ||
*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]] | *Use sample from [[Matt:LabNotes/2014-5-7]] | ||
**A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM | **A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2RevComp in one step | ||
**D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM | **D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM dcProbe2RevComp after | ||
**D5: Negative Ctrl (1uM | **D5: Negative Ctrl (1uM dcProbe2RevComp) | ||
===Procedure=== | ===Procedure=== | ||
*Add 100ul of ppMALAT1 + dcProbe2-RevComp @45C for 24hrs | *Add 100ul of ppMALAT1 + dcProbe2-RevComp @45C for 24hrs | ||
'''A3-A5''' | '''A3-A5''' | ||
{| {{table}} | {| {{table}} | ||
Line 17: | Line 20: | ||
| 2uM ppMALAT1||1.5 | | 2uM ppMALAT1||1.5 | ||
|- | |- | ||
| 100uM | | 100uM dcProbe2RevComp||3 | ||
|- | |- | ||
| H2O||265.5 | | H2O||265.5 | ||
Line 51: | Line 54: | ||
| Total||100 | | 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=== | |||
*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 | 600px]]<br> | |||
'''Fraction of rolonies with hybridized padlock probes'''<br> | |||
[[File:Hybridized_PP_Ratio.JPG | 600px]] | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Position''' | |||
| align="center" style="background:#f0f0f0;"|'''1st Rolonies''' | |||
| align="center" style="background:#f0f0f0;"|'''PISA7 Rolonies''' | |||
| align="center" style="background:#f0f0f0;"|'''PISA7 Overlap''' | |||
| align="center" style="background:#f0f0f0;"|'''bwlabel Rolonies''' | |||
| align="center" style="background:#f0f0f0;"|'''PISA7 Ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''Overlap Ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''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=== | |||
*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 |
Latest revision as of 22:03, 28 May 2014
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