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

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