Matt:LabNotes/2014-5-16: Difference between revisions

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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 dcProbe2-RevComp in one step
**A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2RevComp in one step
**D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM dcProbe2-RevComp after
**D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM dcProbe2RevComp after
**D5: Negative Ctrl (1uM dcProbe2-RevComp)
**D5: Negative Ctrl (1uM dcProbe2RevComp)


===Procedure===
===Procedure===
*Add 100ul of ppMALAT1 + dcProbe2-RevComp @45C for 24hrs (6:30p- )
*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 dcProbe2-RevComp||3
| 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