Matt:LabNotes/2016-9-9: Difference between revisions
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>Mzcai (Created page with " =SplintR Ligase Test 2 = *Previous test ==Reference== *V4 and V7 Capture Experiment *[[Matt:LabNotes/2015-3-19|V4 Cap...") |
>Mzcai m (→Conclusion) |
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*Incubate at 37C for 15min | *Incubate at 37C for 15min | ||
*94C for 2min | *94C for 2min | ||
*Enzyme digest template 37C for 1 hr | *Aliquot 10ul from each into separate strip of tubes and put at 4C for 1 hr until qPCR | ||
** | *Enzyme digest template of remaining mixture 37C for 1 hr | ||
** | **1ul Exo I/III (1:1) for DNA | ||
**1ul RNaseH and Riboshredder (1:1) for RNA | |||
***First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder) | ***First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder) | ||
*94C for 2min | *94C for 2min | ||
*Add 1ul Exo I/III (1:1) for unligated padlock probes to ALL tubes | |||
*37C for 1 hr | |||
*94C for 2min | |||
===qPCR=== | ===qPCR=== | ||
====Primers==== | ====Primers==== | ||
Line 164: | Line 168: | ||
| ISB_CA_AR.T3||CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG||Indx3 | | ISB_CA_AR.T3||CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG||Indx3 | ||
|} | |} | ||
====PCR Test for Non-Enzyme Digested Samples==== | ====PCR Test for Non-Enzyme Digested Samples==== | ||
Line 188: | Line 173: | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | | align="center" style="background:#f0f0f0;"|'''1X Volume''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''16X Volume''' | ||
|- | |- | ||
| Captured template||1||0 | | Captured template||1||0 | ||
|- | |- | ||
| 10uM ISB_CA_AF||0.4|| | | 10uM ISB_CA_AF||0.4||6.4 | ||
|- | |- | ||
| 10uM ISB_CA_AR.T1||0.4|| | | 10uM ISB_CA_AR.T1||0.4||6.4 | ||
|- | |- | ||
| 2X KAPA SYBG MM||12.5|| | | 2X KAPA SYBG MM||12.5||200 | ||
|- | |- | ||
| H2O||10.7|| | | H2O||10.7||171.2 | ||
|- | |- | ||
| Total||25|| | | Total||25||384 | ||
|} | |} | ||
*Aliquot 24ul from 18X master mix and add 1ul captured template | *Aliquot 24ul from 18X master mix and add 1ul captured template | ||
Program | Program | ||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s) | 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min | ||
[[ | |||
[[File: | ==Results== | ||
===Before Exo I/III Digest of Unligated Padlock Probes=== | |||
[[File:20160909_SplintRTest2_qPCR_NotDigested.JPG|350px]][[File:20160909_SplintRTest2_qPCR_Digested.JPG|350px]] | |||
====Template Digested Notes==== | |||
*Biggest factor for ligated products is concentration of padlock probes added | |||
**Suggests there is a lot of non-specific annealing between padlock probes at higher concentrations | |||
*Separation between NTC and RNA template samples increases as padlock probe concentration decreases | |||
**30ng samples looked best, with NTC less than NegCtrl | |||
*PosCtrl less ligated products than NegCtrl is an anomaly | |||
**Probably cause: NegCtrl had 2.5X more padlock probes added than PosCtrl | |||
**15min of Ampligase is not enough time for PosCtrl to have a lot of ligation compared to normal protocol of 24hrs | |||
==== | ====Template Not Digested Notes==== | ||
*Theoretically should be the same? Since RNA template cannot be amplified by KAPA polymerase and thermocycling should denature padlock probes from template | |||
**Maybe PCR primers are occupied with template | |||
* | |||
*Separation between sample and NTC for 820ng and 30ng almost non-existent but 150ng still has good separation | |||
**I could see more template leads to PCR primers being occupied on template, which is why 820ng sample amplifies less than NTC but I would then expect 150ng to show more similarity to that than 30ng | |||
[[ | *PosCtrl and NegCtrl look "correct" | ||
[[File: | ===After Exo I/III Digest of Unligated Padlock Probes=== | ||
[[File:20160912_SplintRTest2_qPCR_NotDigested.JPG|350px]][[File:20160912_SplintRTest2_qPCR_Digested.JPG|350px]] | |||
====Template Digested Notes==== | |||
*This is the "correct" experiment | |||
**Everything points to using less padlock probes limits false positives in NTC | |||
*SplintR is much more active than Ampligase | |||
====Template Not Digested Notes==== | |||
*Oddly this shows the ~30nM Padlock Probe concentration of 150ng samples is the sweet spot | |||
**No idea why there is such a large separation if RNA is not digested | |||
== | ==Conclusion== | ||
* | *56.4C annealing temperature might be too low | ||
* | *Ligase time of 15min might be too long at 37C | ||
* | *Biggest factor for ligated products is concentration of padlock probes | ||
- | *Try in situ, hopefully false positive is not an issue by washing away non-annealed padlock probes | ||
*Need to use a stringent wash buffer (30% formamide)? |
Latest revision as of 22:31, 14 September 2016
SplintR Ligase Test 2[edit]
Reference[edit]
Experimental Outline[edit]
- V8 Padlock Probe capture to RNA
- Ran out of V6 probes used last time
- V8 is a subset of V6 probes (targets constitutive exons instead of contigs of exons)
- Quantify captured products by qPCR
- Also adds Illumina sequencing adapters
Sample Groups[edit]
- Use Universal Human Reference RNA (UHRR 740000-41)
- 3 RNA template concentrations for each (30ng, 150ng and 820ng)
- For DNA template positive control only do 1 sample using 300ng gDNA 12878
- NTC - 8.19ng V8 - SplintR
- NTC - 40.9ng V8 - SplintR
- NTC - 221.1ng V8 - SplintR
- 30ng RNA - 8.19ng V8 - SplintR
- 150ng RNA - 40.9ng V8 - SplintR
- 820ng RNA - 221.1ng V8 - SplintR
- PosCtrl: 300ng DNA - 16.38ng V8 - Ampligase
- NegCtrl: 150ng RNA - 40.9ng V8 - Ampligase
- Summary:
- 3 NTC samples with varying amount of padlock probes that matches experimental sample
- 3 experimental samples of 30ng, 150ng, and 820ng UHRR
- 1 PosCtrl that uses Ampligase for V8 to capture 300ng gDNA (876:1 probe:target ratio)
- 1 NegCtrl that uses Ampligase for V8 to capture 150ng UHRR
Experiment[edit]
V8 Padlock Probe Capture[edit]
- Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc)
- DNA is 12878 80.3ng/ul
- V8 padlock probes: 874nM
- Add 5.2ul to samples 3 & 6 and then dilute remaining ~4ul to 19ul final volume
Sample # | RNA (50ng/ul) | DNA (80.3ng/ul) | V8 (42.7ng/ul) | V8 (8.19ng/ul) | 10X SplintR Buffer (*=Ampligase) | H2O | Total |
1 | 0 | 0 | 0 | 1 | 3 | 26 | 30 |
2 | 0 | 0 | 0 | 5 | 3 | 22 | 30 |
3 | 0 | 0 | 5.2 | 0 | 3 | 21.8 | 30 |
4 | 0.6 | 0 | 0 | 1 | 3 | 25.4 | 30 |
5 | 3 | 0 | 0 | 5 | 3 | 19 | 30 |
6 | 16.4 | 0 | 5.2 | 0 | 3 | 5.4 | 30 |
7 | 0 | 3.8 | 0 | 2 | 3* | 21.2 | 30 |
8 | 3 | 0 | 0 | 5 | 3* | 19 | 30 |
- Added 50ul mineral oil on top
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- Add 3ul Enzyme mix prepared on ice!
- Samples 7-8: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
- Samples 1-6: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O
- Incubate at 37C for 15min
- 94C for 2min
- Aliquot 10ul from each into separate strip of tubes and put at 4C for 1 hr until qPCR
- Enzyme digest template of remaining mixture 37C for 1 hr
- 1ul Exo I/III (1:1) for DNA
- 1ul RNaseH and Riboshredder (1:1) for RNA
- First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder)
- 94C for 2min
- Add 1ul Exo I/III (1:1) for unligated padlock probes to ALL tubes
- 37C for 1 hr
- 94C for 2min
qPCR[edit]
Primers[edit]
Primer | Sequence | Index # |
ISB_CA_AF | AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG | |
ISB_CA_AR.T1 | CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG | Indx1 |
ISB_CA_AR.T2 | CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG | Indx2 |
ISB_CA_AR.T3 | CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG | Indx3 |
PCR Test for Non-Enzyme Digested Samples[edit]
Components | 1X Volume | 16X Volume |
Captured template | 1 | 0 |
10uM ISB_CA_AF | 0.4 | 6.4 |
10uM ISB_CA_AR.T1 | 0.4 | 6.4 |
2X KAPA SYBG MM | 12.5 | 200 |
H2O | 10.7 | 171.2 |
Total | 25 | 384 |
- Aliquot 24ul from 18X master mix and add 1ul captured template
Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min
Results[edit]
Before Exo I/III Digest of Unligated Padlock Probes[edit]
File:20160909 SplintRTest2 qPCR NotDigested.JPGFile:20160909 SplintRTest2 qPCR Digested.JPG
Template Digested Notes[edit]
- Biggest factor for ligated products is concentration of padlock probes added
- Suggests there is a lot of non-specific annealing between padlock probes at higher concentrations
- Separation between NTC and RNA template samples increases as padlock probe concentration decreases
- 30ng samples looked best, with NTC less than NegCtrl
- PosCtrl less ligated products than NegCtrl is an anomaly
- Probably cause: NegCtrl had 2.5X more padlock probes added than PosCtrl
- 15min of Ampligase is not enough time for PosCtrl to have a lot of ligation compared to normal protocol of 24hrs
Template Not Digested Notes[edit]
- Theoretically should be the same? Since RNA template cannot be amplified by KAPA polymerase and thermocycling should denature padlock probes from template
- Maybe PCR primers are occupied with template
- Separation between sample and NTC for 820ng and 30ng almost non-existent but 150ng still has good separation
- I could see more template leads to PCR primers being occupied on template, which is why 820ng sample amplifies less than NTC but I would then expect 150ng to show more similarity to that than 30ng
- PosCtrl and NegCtrl look "correct"
After Exo I/III Digest of Unligated Padlock Probes[edit]
File:20160912 SplintRTest2 qPCR NotDigested.JPGFile:20160912 SplintRTest2 qPCR Digested.JPG
Template Digested Notes[edit]
- This is the "correct" experiment
- Everything points to using less padlock probes limits false positives in NTC
- SplintR is much more active than Ampligase
Template Not Digested Notes[edit]
- Oddly this shows the ~30nM Padlock Probe concentration of 150ng samples is the sweet spot
- No idea why there is such a large separation if RNA is not digested
Conclusion[edit]
- 56.4C annealing temperature might be too low
- Ligase time of 15min might be too long at 37C
- Biggest factor for ligated products is concentration of padlock probes
- Try in situ, hopefully false positive is not an issue by washing away non-annealed padlock probes
- Need to use a stringent wash buffer (30% formamide)?