Blue:RNA-Seq Experiments:10232013: Difference between revisions
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**1ng unfragmented UHRR | **1ng unfragmented UHRR | ||
**1ng UHRR fragmented in RNAseIII buffer | **1ng UHRR fragmented in RNAseIII buffer | ||
***Without PAP | ***Without PAP (no heat inactivation) | ||
**1ng UHRR fragmented in PAP Buffer | **1ng UHRR fragmented in PAP Buffer | ||
***Without PAP | ***Without PAP | ||
***With PAP | ***With PAP | ||
****1mM ATP | ****1mM ATP - 10 min PAP | ||
****1mM ATP/0.1mM 3'd-ATP | ****1mM ATP - 30 min PAP | ||
**1uM TSO in PAP buffer | ****1mM ATP/0.5mM 3'd-ATP - 10 min PAP | ||
****1mM ATP/0.5mM 3'd-ATP - 30 min PAP | |||
**1uM TSO in PAP buffer (1mM ATP) | |||
***Without PAP | ***Without PAP | ||
***With PAP | ***With PAP | ||
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:::''Incubate @ 37C | :::''Incubate @ 37C 10 min or 30 min'' | ||
:::''Incubate @ 65C 10 min'' | :::''Incubate @ 65C 10 min'' | ||
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|5x PAP Buffer||||||||||0.72ul | |5x PAP Buffer||||||||||0.72ul | ||
|- | |- | ||
|MnCl2 (Ambion)||||||||||0. | |MnCl2 (Ambion)||||||||||0.32ul | ||
|- | |- | ||
|1mM ATP (or mix)||||||||||0.25ul | |1mM ATP (or mix)||||||||||0.25ul | ||
|- | |- | ||
|0. | |0.4U/ul PAP ||||||||||0.25ul | ||
|- | |- | ||
|dH20||||||||||0. | |dH20||||||||||0.46ul | ||
|- | |- | ||
|Total||||||||||4ul | |Total||||||||||4ul |
Revision as of 19:25, 25 October 2013
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:
Result: Ambion PAP shows best PolyA activity. Both Ambion and Enzymatics PAP creates a smear up to and exceeding 300bp. Affymetrix PAP added way too many A's.
Tested minimal number of PAP units required:
Result: Significant drop off of activity for Ambion below 0.4U - no effect on number of A's added. Affymetrix PAP cannot be diluted to low unit volumes.
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
Result: Progressive restriction of Ambion PAP activity was seen with increasing 3'dATP. Affymetrix PAP was completely inhibited by the lowest amount of 3'dATP added.
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
Result: Unlike in commercial buffer, Ambion PAP and Enzymatics PAP were not inhibited by 3'dATP in totoRNAseq buffer. 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...]
totoRNAseq buffer [8.75mM total 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 (no heat inactivation)
- 1ng UHRR fragmented in PAP Buffer
- Without PAP
- With PAP
- 1mM ATP - 10 min PAP
- 1mM ATP - 30 min PAP
- 1mM ATP/0.5mM 3'd-ATP - 10 min PAP
- 1mM ATP/0.5mM 3'd-ATP - 30 min PAP
- 1uM TSO in PAP buffer (1mM ATP)
- Without PAP
- With PAP
- With PAP (reaction pre-heat inactivated without TSO 10 min @ 65C)
- With PAP (reaction pre-heat inactivated without TSO 10 min @ 72C)
- With PAP (reaction pre-heat inactivated without TSO 10 min @ 75C)
- RNA FRAGMENTATION
Volume UHRR 2ng/ul 0.5ul 10x RNaseIII Buffer 0.2ul or 5x PAP Buffer (Ambion) 0.08ul 25mM MnCl2 (Ambion) 0.08ul RNase III 0.08ul dH20 1.22ul (RIII) or 1.26ul (Ambion) Total 2ul
- Incubate @ 37C 10 min or 30 min
- Incubate @ 65C 10 min
- 3' Tailing
Volume fRNA 2ul 5x PAP Buffer 0.72ul MnCl2 (Ambion) 0.32ul 1mM ATP (or mix) 0.25ul 0.4U/ul PAP 0.25ul dH20 0.46ul Total 4ul
- Incubate @ 37C 10 min