Editing
Blue:RNA-Seq Experiments:10232013
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
== 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''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |1uM TSO.r04||||||||||1ul |- |dH20||||||||||6ul |- |10x PAP buffer||||||||||1ul |- |10mM ATP||||||||||1ul |- |0.5U/ul PAP (1:10 Dil)||||||||||1ul |- |} :'''Affymetrix 3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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: [[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]] 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 [[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 == *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 :'''totoRNAseq Reaction Buffer''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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 |- |} [[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...
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Template used on this page:
Template:Table
(
edit
)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information