Blue:RNA-Seq Experiments:10232013

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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:


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[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


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[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


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


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
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


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[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


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...