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==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]] **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=== *Add 100ul of ppMALAT1 + dcProbe2-RevComp @45C for 24hrs '''A3-A5''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||30 |- | 2uM ppMALAT1||1.5 |- | 100uM dcProbe2RevComp||3 |- | H2O||265.5 |- | Total||300 |} '''D3&D4''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 2uM ppMALAT1||1 |- | H2O||179 |- | Total||200 |} '''D5''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''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=== *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
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