Matt:LabNotes/2014-1-31
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Shifted FISSEQ vs FISSEQ_Adpt Signal Count in Same Wells
- Previously compared the two dyes in adjacent wells Matt:LabNotes/2014-1-28
- Here I strip FISSEQ_Adpt from before and add Shifted FISSEQ to column 9
- Put Shifted FISSEQ_2ndRCAprimerDye in columns 4 and 9
- Put RAB7A_1stRolonyFISH in columns 3 and 10
Well | RT primer | Cy3 | Shifted | % of Cy3 |
C9 | 220nt | 711 | 129 | 18.14 |
D9 | 300nt | 357 | 203 | 56.86 |
E9 | 510nt | 4449 | 842 | 18.923 |
F9 | 1000nt | 13295 | 14654 | 110.22 |
Average % of Cy3: | 51.04 |
Conclusions
- If we take the very rough estimate that 51% of primary rolonies are hybridized by Shifted FISSEQ dye
- Previously when we hybridized ppMALAT1 and found 59% of primary rolonies are MALAT1, then the true number of MALAT1 rolonies is 59-51 = ~8%
RT_RAB7A Primers and the Number of RAB7A Primary Rolonies
- Using RAB7A_1stRolonyFISH (ATTO550) in column 10
Well | RT primer | Count |
C10 | 220nt | 24 |
D10 | 300nt | 24 |
E10 | 510nt | 13 |
F10 | 1000nt | 35 |
F3 | RandomHex | 5 |
Including Hosuk's Results Hosuk:LabNotes/2013-12-30#Result
- Combining my results (Matt:LabNotes/2014-1-28#Results) with Hosuk's
- Hosuk didn't see any rolonies using RAB7A_1stRolonyFISH
File:RTprimerRolonyCountComparison.JPG
Conclusions
- The different RT primer sequences definitely have a consistent effect on number of primary rolonies that can be made
- Most likely the 10nt primer sequence for 510nt and 1000nt matches some very high abundance transcripts (explore whether it matches ribosomal RNA here: Matt:LabNotes/2014-2-3
- Since most primary rolonies aren't RAB7A no conclusions can be drawn whether distance from TSS affects CircLigase efficiency
- Although there's only one set of RAB7A_1stRolonyFISH dye samples and the primary rolony counts are low, using targeted primers could still help increase yield of specific gene primary rolonies