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== 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...
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