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==Agi26k0gap Secondary Rolonies Mattek Dish== *Since we now have a [[Matt:LabNotes/2014-2-25#Procedure|successful procedure for generating Secondary Rolonies]] *Since we have optimized PGP1f sample prep by culturing without EGF ([[Hosuk:LabNotes/2014-3-3]]) **In Harvard Science paper (Lee, J. et al), this culture condition resulted in more mRNA and less rRNA **Also microarray data on GEO shows Agi26k0gap probe set targets many high expression transcripts in PGP1f cells [[Matt:LabNotes/2014-3-7]] *Try to generate highly multiplexed 2nd rolonies with 12k padlock probeset **30.3 pmole of Agi26k0gap prepared [[Matt:LabNotes/2014-3-5|LabNotes/2014-3-5]] ***30.3 pmole / 200ul = 0.1515 pmole/ul = 0.1515 umole/L = 151.5nM ****151.5nM/12,000 probes = 0.0126 nM = 12.6 pM ===Procedure=== *Using Mattek dish #2 from [[Hosuk:LabNotes/2014-3-3]] that was fixed in 10% formalin ====Padlock Probe Capture==== #Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min #Wash with 1X PBS twice #Anneal Padlock Probes @45C for 28hrs (Put in at 11:30a) #*1X Ampligase Buffer #*~12.6nM of each padlock probe #*0.5 U/ul Ampligase #*Prepared everything on ice and then pre-heated to 75C for 5 min before adding {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 30.3 pmole Agi26k0gap||58 |- | 5U/ul Ampligase||20 |- | H2O||102 |- | Total||200 |} ====Secondary RCA==== #Aspirate but don't wash #'''Pre-annealing the FISSEQ_ppRCA primer''' for the rolling circle amplification reaction. ##1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of '''2xSSC/30% formamide''' ##Preheat primer at 60C (Tm = 55C when '''not''' in 30% formamide) ##Add 200ul and incubate at 45C for 15min. ##Aspirate, and wash using 2x SSC twice ##Aspirate, and wash using 0.1x SSC twice #Prepare the rolling circle amplification reaction mixtures on ice. ##171 ul ddH2O, ##20 ul 10x Phi29 buffer, ##2 ul 25 mM dNTP, ##4 ul 2 mM aminoallyl dUTP ##3 ul Phi29 DNA polymerase (Epicentre, low concentration) ##*200ul Total Volume #Add and incubate 20hrs at 30C. (Put in at 4:15pm 3/11/14) #Wash 1X PBS once #Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr #Wash 1X PBS twice #Add 200ul 1M Tris pH 8.0 @RT for 30min #Wash 1X PBS twice ===Detection=== ====Initial Imaging==== *3/12/2014 *Check to see if 2nd rolony generation was successful by detecting a large fraction **Use 1uM dcProbe0-Cy3 and 1uM dcProbe0-FAM in 30% formamide + 2X SSC ***This should detect ~1/2 of all 2nd rolonies ===Results=== ====Initial Imaging==== *Using PISA7 Paramters: 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 lower (variable) *First used PISA to examine the images with varying bkgmult lower (range: 2-9) {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Bkgmult''' | align="center" style="background:#f0f0f0;"|'''Rolony Count''' | align="center" style="background:#f0f0f0;"|'''Notes''' |- | NoDye_G680_Cy3||7||116|| |- | NoDye_G680_Cy3||9||31|| |- | NoDye_L2%_G680_FAM||7||202||picked up mostly nuclei |- | NoDye_L2%_G680_FAM||9||7|| |- | dcProbe0-Cy3_G550||7||178|| |- | dcProbe0-Cy3_G550||9||162|| |- | dcProbe0-Cy3_G550||5||208|| |- | dcProbe0-Cy3_G550||3||265||decreasing bkgmult detects more "true" features without significantly counting artifacts |- | dcProbe0-Cy3_G550||2||278||doesn't detect anymore "true" features, just adds noise |- | dcProbe0-Cy3_G680||7||544|| |- | dcProbe0-Cy3_G680||9||489|| |- | dcProbe0-FAM_L5%_G550||7||129|| |- | dcProbe0-FAM_L5%_G550||3||209|| |- | dcProbe0-FAM_L5%_G550||2||245||still misses many while counting artifacts |- | dcProbe0-FAM_G550||7||52|| |- | dcProbe0-FAM_G550||5||68|| |- | dcProbe0-FAM_G550||3||108||even lowering it to 3 still won't count obvious features |- | dcProbe0-FAM_G600||7||313|| |- | dcProbe0-FAM_G600||5||364||good |- | dcProbe0-FAM_G680||7||519|| |- | dcProbe0-FAM_G680||9||375||not bad, pretty similar to G600 bkgmult5 |} '''Count Cells in Field of View''' *NoDye_L2%_G680_FAM had nuclei with high intensity fluorescence relative to background, which was really obvious after processing in PISA so counted nuclei in that image **110 Nuclei/Cells counted [[File:Agi26k0gap_PGP1f_InitialDetect_NucleiCount110.png|450px]] '''Best Images and PISA Settings''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Image Name''' | align="center" style="background:#f0f0f0;"|'''Image''' | align="center" style="background:#f0f0f0;"|'''Feature Count''' |- | Cy3 Background||NoDye_G680_Cy3_Bkgmult9||[[File:NoDye_G680_Cy3_ch00_PISA7Overlay_Bkgmult9.png|450px]]||31 |- | FAM Background||NoDye_L2%_G680_FAM_Bkgmult9||[[File:NoDye_L2%25_G680_FAM_ch00_PISA7Overlay_Bkgmult9.png|450px]]||7 |- | dcProbe0-Cy3||dcProbe0Cy3_G550_Bkgmult3||[[File:DcProbe0Cy3_G550_Cy3_ch00_PISA7Overlay_Bkgmult3.png|450px]]||265 |- | dcProbe0-FAM||dcProbe0FAM_G600_Bkgmult5||[[File:DcProbe0FAM_600_FAM_ch00_PISA7Overlay_Bkgmult5.png|450px]]||364 |} '''Removing Background''' *'''dcProbe0-Cy3''' Ran PISA on NoDye_G680_Cy3_Bkgmult9 and dcProbe0Cy3_G550_Bkgmult3 Saved bw images in workspace as backgroundCy3 and dcProbe0Cy3 countOverlappedPoloniesv2(backgroundCy3,dcProbe0Cy3) ans = 0 comp_img1=zeros(xsize,ysize,3); comp_img1(:,:,1)=dcProbe0Cy3; comp_img1(:,:,2)=backgroundCy3; imwrite(comp_img1,'dcProbe0Cy3vsbackgroundCy3.png'); [[File:DcProbe0Cy3vsbackgroundCy3.png|450px]] *'''dcProbe0-FAM''' Ran PISA on NoDye_L2%_G680_FAM_Bkgmult9 and dcProbe0FAM_G600_Bkgmult5 Saved bw images in workspace as backgroundFAM and dcProbe0FAM countOverlappedPoloniesv2(backgroundFAM,dcProbe0FAM) ans = 0 comp_img1=zeros(xsize,ysize,3); comp_img1(:,:,1)=dcProbe0Cy3; comp_img1(:,:,2)=backgroundCy3; imwrite(comp_img1,'dcProbe0Cy3vsbackgroundCy3.png'); [[File:DcProbe0FAMvsbackgroundFAM.png|450px]] *Conclusion: background is not a factor '''Specificity of dcProbe0-Cy3 and dcProbe0-FAM''' *Is there overlap between the two? (There shouldn't be, they should be exclusive) countOverlappedPoloniesv2(dcProbe0Cy3,dcProbe0FAM) ans = 1 [[File:DcProbe0Cy3vsdcProbe0FAM.png|450px]] *The only overlap is in the bottom right corner and looks more likely due to nearby rolonies than non-specific binding of both dyes to some structure **Red is dcProbe0-Cy3 and Green is dcProbe0-FAM '''Overlay BrightField'''<br> [[File:DcProbe0Cy3vsdcProbe0FAM_BF.png|650px]] ===Conclusion=== ====Initial Imaging==== *Background signal did not play a factor since none overlapped with detected features during dcProbe0-Cy3 and dcProbe-FAM **We can say every feature we see is specific to 2nd rolony *dcProbe0-Cy3 and dcProbe0-FAM showed essentially no overlap, which gives us confidence these are true signals **Next time, we should be able to detect dcProbe1, dcProbe2, etc. and expect to see a fraction overlap with these *dcProbe0-Cy3 had 265 rolonies (~2.4/cell) *dcProbe0-FAM had 364 rolonies (~3.3/cell) *dcProbe0-Cy3(3641 probes) + dcProbe0-FAM (4097 probes) = 7,738 probes/12,964 total probes in Agi26k0gap = 60% **If these two dyes which can detect 60% of probes detected 629 2nd rolonies then we can approximate the total number of 2nd rolonies in this field of view = ~1050 *There are ~9.5 2nd rolonies/cell
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