Matt:LabNotes/2014-1-31: Difference between revisions

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>Mzcai
 
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===Testing===
===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 in columns 4 and 9
*Put Shifted FISSEQ_2ndRCAprimerDye in columns 4 and 9
*Put RAB7A_1stRolonyFISH in columns 3 and 10
*Put RAB7A_1stRolonyFISH in columns 3 and 10
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''RT primer'''
| align="center" style="background:#f0f0f0;"|'''Cy3'''
| align="center" style="background:#f0f0f0;"|'''Shifted'''
| align="center" style="background:#f0f0f0;"|'''% 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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''RT primer'''
| align="center" style="background:#f0f0f0;"|'''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

Latest revision as of 23:47, 6 February 2014

Shifted FISSEQ vs FISSEQ_Adpt Signal Count in Same Wells[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

File:RTprimerRolonyCountComparison.JPG

Conclusions[edit]

  • 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