Matt:LabNotes/2014-3-4
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3 Genes Secondary Rolonies Mattek Dish
- Hosuk got much more primary rolonies (incl. ACTB and RAB7A) by culturing PGP1F without EGF (Hosuk:LabNotes/2014-3-3)
- Since Matt:LabNotes/2014-2-25#Detection resulted in many MALAT1 secondary rolonies but few ACTB or RAB7A
- This is possibly due to sample having very few ACTB or RAB7A
- Also possible the dyes we got from Illumina (dcProbe0-Cy3 and dcProbe1-Cy3) have somehow degraded since MALAT1 detection was using a new dye-probe from IDT
Procedure
- Using Mattek dish from Hosuk:LabNotes/2014-3-3 that was fixed in 4% formalin
- 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 24hrs (Put in at noon, some bubbles and hard to keep on middle glass)
- 1X Ampligase Buffer
- 2uM of each padlock probe
- 0.5 U/ul Ampligase
Component | Volume |
10X Ampligase Buffer | 20 |
200uM ppMALAT1 | 2 |
200uM ppACTB | 2 |
200uM ppRAB7A | 2 |
5U/ul Ampligase | 20 |
H2O | 154 |
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 1pm 3/5/14)
- 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 1hr
- Wash 1X PBS twice
Detection
- Make 100ul of 1uM dcProbe0-Cy3, dcProbe1-Cy3, and dcProbe2-Cy3 in 30% formamide and 2X SSC
- dcProbe0 and dcProbe1 are from Illumina, dcProbe2 is from IDT
- Need to order more dcProbe1-Cy3
- dcProbe0 and dcProbe1 are from Illumina, dcProbe2 is from IDT
- Checked dcProbe0
- Strip
- Checked dcProbe1
- Strip
- Checked dcProbe2
- Dust fell into dish? Lots of non-specific binding so can't see secondary rolonies anymore
Results
- 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 (7)
dcProbe0
- ACTB 2nd Rolonies
- Pos1: 108
- Pos2:
Gain 680 | Gain 620 |
130 2nd Rolonies | 244 2nd Rolonies |
File:Pos2 noEGF dcProbe0 680 Cy3 ch00 MIP PISA7Overlay.png | File:Pos2 noEGF dcProbe0 620 Cy3 ch00 MIP PISA7Overlay.png |
dcProbe1
- RAB7A 2nd Rolonies
- Pos2:
Gain 680 | Gain 620 |
595 2nd Rolonies | 779 2nd Rolonies |
File:Pos2 noEGF dcProbe1 680 Cy3 ch00 MIP PISA7Overlay.png | File:Pos2 noEGF dcProbe1 620 Cy3 ch00 MIP PISA7Overlay.png |
dcProbe2
- Didn't analyze because of dust
Conclusion
- The goal was to see if we could get ACTB and RAB7A 2nd Rolonies
- Goal was successful
- There is no problem with dye-probes from Illumina or ppACTB/ppRAB7A
- Previously the lack of ACTB and RAB7A 2nd Rolonies must have been due to low amount of mRNA in fixed sample
- Goal was successful