Blue:RNA-Seq Experiments:10232013
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Purpose
- Test PAP Activity from different companies:
- Ambion
- Enzymatics (used up to this point)
- Affymetrix
- Test Blocking of PAP activity using Cordycepin-ATP
Commercial Protocols
- Ambion 3' Tailing
Volume 1uM TSO.r04 1ul dH20 4.6ul 5x E-PAP buffer 2ul 25mM MnCl2 1ul 10mM ATP 1ul 2U/ul PAP 0.4ul
- Enzymatics 3' Tailing
Volume 1uM TSO.r04 1ul dH20 6ul 10x PAP buffer 1ul 10mM ATP 1ul 0.5U/ul PAP (1:10 Dil) 1ul
- Affymetrix 3' Tailing
Volume 1uM TSO.r04 1ul dH20 5ul 5x PAP buffer 2ul 10mM ATP 1ul 60U/ul PAP (1:10 Dil) 1ul
- Incubate @ 37C 0, 5, 10, 15, 20 min
- Transfer to Ice
- Run 5ul on urea gel:
Poly A Limiting Tests
- 3'dATP Blocking
- Test:
- Ambion PAP (0.4U) with 3'dATP
- 1mM ATP/0mM 3'ATP
- 1mM ATP/0.01mM 3'ATP
- 1mM ATP/0.05mM 3'ATP
- 1mM ATP/0.1mM 3'ATP
- 1mM ATP/0.5mM 3'ATP
- 0mM ATP/1mM 3'ATP
- Affymetrix PAP (60U)with 3'dATP
- 1mM ATP/0mM 3'ATP
- 1mM ATP/0.01mM 3'ATP
- 1mM ATP/0.05mM 3'ATP
- 1mM ATP/0.1mM 3'ATP
- 1mM ATP/0.5mM 3'ATP
- 0mM ATP/1mM 3'ATP
- Ambion PAP (0.4U) with 3'dATP
Adaptation to totoRNAseq
- Test:
- Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer
- Commercial mix Ambion (see above)
- 10mM ATP
- 1mM ATP
- 1mM ATP/0.1mM 3' d-ATP
- 1mM ATP/0.5mM 3' d-ATP
- totoRNAseq mix Ambion (below)
- 10mM ATP
- 1mM ATP
- 1mM ATP/0.1mM 3' d-ATP
- 1mM ATP/0.5mM 3' d-ATP
- totoRNAseq mix Enzymatics (below)
- 10mM ATP
- 1mM ATP
- 1mM ATP/0.1mM 3' d-ATP
- 1mM ATP/0.5mM 3' d-ATP
- Commercial mix Ambion (see above)
- Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer
- totoRNAseq Reaction Buffer
Volume 1uM TSO.r04 1ul dH20 6.4ul 5x RT buffer 1.25ul 10x RnaseIII buffer 0.5ul 1mM ATP or 10mM ATP 0.625ul 0.5U PAP 0.25ul
Note: totoRNAseq buffer has too high of Mg levels - uncontrolled PAP activity
Ambion 1x buffer [50mM Tris; 0.25M NaCl2; 2.5mM MnCl2]
RNaseIII 1x buffer [10mM Tris; 10mM MgCl2; 1mM DTT; 60mM NaCl]
MMLV RT Buffer [30mM Mgcl2...]
- Test:
- Ambion PAP without RT buffer
- Commercial mix Ambion (see above)
- 10mM ATP
- 1mM ATP
- 1mM ATP/0.1mM 3' d-ATP
- 1mM ATP/0.5mM 3' d-ATP
- totoRNAseq mix -RT (below)
- 10mM ATP
- 1mM ATP
- 1mM ATP/0.1mM 3' d-ATP
- 1mM ATP/0.5mM 3' d-ATP
- totoRNAseq mix -RT +NaCl (below)
- 10mM ATP
- 1mM ATP
- 1mM ATP/0.1mM 3' d-ATP
- 1mM ATP/0.5mM 3' d-ATP
- Commercial mix Ambion (see above)
- Ambion PAP without RT buffer
- totoRNAseq Reaction Buffer
Volume 1uM TSO.r04 1ul dH20 7.625ul (6.525 + 1.1ul 2M NaCl) 10x RnaseIII buffer 0.5ul ATP 0.625ul 0.5U PAP 0.25ul
Note: Best PAP activity is in the Commercial buffer conditions - therefore may need to determine whether RNAseIII can still digest under PAP buffer conditions...
Testing RNAaseIII Activity in PAP Buffer
Ambion 1x buffer [50mM Tris; 0.25M NaCl2; 2.5mM MnCl2]
RNaseIII 1x buffer [10mM Tris; 10mM MgCl2; 1mM DTT; 60mM NaCl]
- Test:
- 1ng unfragmented UHRR
- 1ng UHRR fragmented in RNAseIII buffer
- Without PAP
- 1ng UHRR fragmented in PAP Buffer
- Without PAP
- With PAP
- 1mM ATP
- 1mM ATP/0.1mM 3'd-ATP
- RNA FRAGMENTATION
Volume UHRR 2ng/ul 0.5ul 10x RNaseIII Buffer 0.2ul or 5x PAP Buffer (Ambion) 0.08ul MnCl2 (Ambion) 0.2ul RNase III 0.08ul dH20 1.22ul (RIII) or 1.14ul (Ambion) Total 2ul
- Incubate @ 37C 5-10 min
- Incubate @ 65C 10 min
- 3' Tailing
Volume fRNA 2ul 5x PAP Buffer 0.72ul MnCl2 (Ambion) 0.2ul 1mM ATP (or mix) 0.25ul 0.5U/ul PAP (1:10d in H2O) 0.25ul dH20 0.58ul Total 4ul
- Incubate @ 37C 10 min