Blue:RNA-Seq Experiments:10232013: Difference between revisions
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[[File:10232013_gel1.jpg]] | [[File:10232013_gel1.jpg]] | ||
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: | |||
[[File:10232013_gel2.jpg]] | [[File:10232013_gel2.jpg]] | ||
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 == | == Poly A Limiting Tests == | ||
Line 106: | Line 113: | ||
[[File:10232013_gel3.jpg]] | |||
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 == | == Adaptation to totoRNAseq == | ||
*Test: | *Test: | ||
**Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer | **Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer | ||
***Commercial mix (see above) | ***Commercial mix Ambion (see above) | ||
***totoRNAseq mix | ****10mM ATP | ||
***totoRNAseq mix | ****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 | |||
:'''totoRNAseq Reaction Buffer''' | |||
::{| {{table}} | ::{| {{table}} | ||
Line 126: | Line 148: | ||
|1uM TSO.r04||||||||||1ul | |1uM TSO.r04||||||||||1ul | ||
|- | |- | ||
|dH20|||||||||| | |dH20||||||||||6.4ul | ||
|- | |- | ||
|5x RT buffer||||||||||1.25ul | |5x RT buffer||||||||||1.25ul | ||
Line 134: | Line 156: | ||
|1mM ATP or 10mM ATP||||||||||0.625ul | |1mM ATP or 10mM ATP||||||||||0.625ul | ||
|- | |- | ||
|0.5U PAP|||||||||| | |0.5U PAP||||||||||0.25ul | ||
|- | |- | ||
|} | |} | ||
[[File:10232013_gel4.jpg]] | |||
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 | |||
:'''totoRNAseq Reaction Buffer''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|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 | |||
|- | |||
|} | |||
[[File:10232013_gel5.jpg]] | |||
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''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|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''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|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'' | |||
[[File:10232013_gel6.jpg]] | |||
Results: Unfortunately 1ng RNA is not visible... Can see that the RNaseIII fragmentation/PAP conditions do allow blockage by 3'dATP. Extended PAP treatment only resulted in a minor increase in smear intensity. TSO polyA tailing was blocked by heat inactivation of PAP at all temperatures tested. The smear for fragmented RNA and TSO are remarkable similar... what is actually being polyA tailed in the RNA? | |||
[[File:10232013_gel7.jpg]] | |||
Results: Comparison of 10ng unfragmented RNA with 1uM TSO for Ambion PAP treatment to see if there is a bias for smaller RNA. RNA alone shows smear that must have come from LMW RNA fragments that weren't even visible in the PAP untreated lane, indicating that there might be a bias for smaller RNA fragments. Combining TSO with RNA does not show much - can see both a higher and a lower smear for each, no real preference... Results indicate that it might be best to reduce RNA pool to a more consistent and uniformly sized LMW population to overcome potential bias... |
Latest revision as of 22:41, 25 October 2013
Purpose[edit]
- Test PAP Activity from different companies:
- Ambion
- Enzymatics (used up to this point)
- Affymetrix
- Test Blocking of PAP activity using Cordycepin-ATP
Commercial Protocols[edit]
- 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[edit]
- 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[edit]
- 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[edit]
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
Results: Unfortunately 1ng RNA is not visible... Can see that the RNaseIII fragmentation/PAP conditions do allow blockage by 3'dATP. Extended PAP treatment only resulted in a minor increase in smear intensity. TSO polyA tailing was blocked by heat inactivation of PAP at all temperatures tested. The smear for fragmented RNA and TSO are remarkable similar... what is actually being polyA tailed in the RNA?
Results: Comparison of 10ng unfragmented RNA with 1uM TSO for Ambion PAP treatment to see if there is a bias for smaller RNA. RNA alone shows smear that must have come from LMW RNA fragments that weren't even visible in the PAP untreated lane, indicating that there might be a bias for smaller RNA fragments. Combining TSO with RNA does not show much - can see both a higher and a lower smear for each, no real preference... Results indicate that it might be best to reduce RNA pool to a more consistent and uniformly sized LMW population to overcome potential bias...