Matt:LabNotes/2014-1-31: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''% of Cy3''' | | align="center" style="background:#f0f0f0;"|'''% of Cy3''' | ||
|- | |- | ||
| C9||220nt||711||129|| | | C9||220nt||711||129||18.14 | ||
|- | |- | ||
| D9||300nt||357||203|| | | D9||300nt||357||203||56.86 | ||
|- | |- | ||
| E9||510nt||4449||842|| | | E9||510nt||4449||842||18.923 | ||
|- | |- | ||
| F9||1000nt||13295||14654|| | | F9||1000nt||13295||14654||110.22 | ||
|- | |- | ||
| ||||||Average % of Cy3:|| | | ||||||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=== | ===RT_RAB7A Primers and the Number of RAB7A Primary Rolonies=== | ||
*Using RAB7A_1stRolonyFISH (ATTO550) | *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]
- 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[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