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
 
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Line 143: Line 143:
*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
**2ul Exo I/III (1:1) for DNA
*Enzyme digest template of remaining mixture 37C for 1 hr
**2ul RNaseH and Riboshredder (1:1) for RNA
**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
|}
|}
<!--
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Index'''
| align="center" style="background:#f0f0f0;"|'''Forward Primer'''
| align="center" style="background:#f0f0f0;"|'''Reverse Primer'''
|-
| 1||1||ISB_CA_AF||ISB_CA_AR.T1
|-
| 2||2||ISB_CA_AF||ISB_CA_AR.T2
|-
| 3||3||ISB_CA_AF||ISB_CA_AR.T3
|-
| 4||1||ISB_CA_AF||ISB_CA_AR.T1
|-
| 5||2||ISB_CA_AF||ISB_CA_AR.T2
|-
| 6||3||ISB_CA_AF||ISB_CA_AR.T3
|}-->


====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;"|'''18X Volume'''
| align="center" style="background:#f0f0f0;"|'''16X Volume'''
|-
|-
| Captured template||1||0
| Captured template||1||0
|-
|-
| 10uM ISB_CA_AF||0.4||7.2
| 10uM ISB_CA_AF||0.4||6.4
|-
|-
| 10uM ISB_CA_AR.T1||0.4||7.2
| 10uM ISB_CA_AR.T1||0.4||6.4
|-
|-
| 2X KAPA SYBG MM||12.5||225
| 2X KAPA SYBG MM||12.5||200
|-
|-
| H2O||10.7||192.6
| H2O||10.7||171.2
|-
|-
| Total||25||432
| 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)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min
[[Media:SplintR_Ligase_Test_qPCR_-_No_Enzyme_Digest.xlsx|Excel sheet with full PCR data]]<br>
 
[[File:SplintR_Ligase_Test_qPCR_-_No_Enzyme_Digest.JPG|450px]]
==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


====PCR Test for Enzyme Digested Samples====
====Template Not Digested Notes====
{| {{table}}
*Theoretically should be the same? Since RNA template cannot be amplified by KAPA polymerase and thermocycling should denature padlock probes from template
| align="center" style="background:#f0f0f0;"|'''Components'''
**Maybe PCR primers are occupied with template
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
| align="center" style="background:#f0f0f0;"|'''18X Volume'''
|-
| Captured template||1||0
|-
| 10uM ISB_CA_AF||0.4||7.2
|-
| 10uM ISB_CA_AR.T1||0.4||7.2
|-
| 2X KAPA SYBG MM||12.5||225
|-
| H2O||10.7||192.6
|-
| Total||25||432
|}
*Aliquot 24ul from 18X master mix and add 1ul captured template


  Program
*Separation between sample and NTC for 820ng and 30ng almost non-existent but 150ng still has good separation
  98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
**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
[[Media:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.xlsx|Excel sheet with full PCR data]]<br>
*PosCtrl and NegCtrl look "correct"
[[File:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.JPG|450px]]
===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


==Results & Conclusion==
==Conclusion==
*Pos Ctrl (V6 probes capture gDNA with Ampligase) has low amount of ligated padlock probes
*56.4C annealing temperature might be too low
**High Ct in PCR curve
*Ligase time of 15min might be too long at 37C
**I guess 300ng gDNA has fewer targets than 30ng RNA
*Biggest factor for ligated products is concentration of padlock probes
**But same low level as Neg Ctrl (Ampligase used on DNA/RNA hybrid)
*NTC (V6 captures '''nothing''' with SplintR) has same amount of ligated padlock probes as Experimental samples (V6 captures RNA with SplintR)
**suggests SplintR doesn't need RNA splint to ligate DNA


*CONCLUSION: Need to check literature and/or call NEB but it seems SplintR has high ssDNA ligase activity, making it unusable for high specificity padlock probe applications
**Checked and there is no reported ssDNA ligase activity, therefore it must be probes annealing to other probes
**"Template-independent ligation"


-->
*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]

  1. 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)
  2. 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
  1. NTC - 8.19ng V8 - SplintR
  2. NTC - 40.9ng V8 - SplintR
  3. NTC - 221.1ng V8 - SplintR
  4. 30ng RNA - 8.19ng V8 - SplintR
  5. 150ng RNA - 40.9ng V8 - SplintR
  6. 820ng RNA - 221.1ng V8 - SplintR
  7. PosCtrl: 300ng DNA - 16.38ng V8 - Ampligase
  8. 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)?