Blue:RNA-Seq Experiments:10232013: Difference between revisions

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| or 5x PAP Buffer (Ambion)||||||||||0.08ul
| or 5x PAP Buffer (Ambion)||||||||||0.08ul
|-
|-
| MnCl2 (Ambion)||||||||||0.2ul
| 25mM MnCl2 (Ambion)||||||||||0.08ul
|-
|-
|RNase III||||||||||0.08ul  
|RNase III||||||||||0.08ul  
|-
|-
|dH20||||||||||1.22ul (RIII) or 1.14ul (Ambion)
|dH20||||||||||1.22ul (RIII) or 1.26ul (Ambion)
|-
|-
|Total||||||||||2ul
|Total||||||||||2ul

Revision as of 17:50, 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:


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
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

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
    • 1ng UHRR fragmented in PAP Buffer
      • Without PAP
      • With PAP
        • 1mM ATP
        • 1mM ATP/0.1mM 3'd-ATP


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 5-10 min
Incubate @ 65C 10 min



3' Tailing
Volume
fRNA 2ul
5x PAP Buffer 0.72ul
MnCl2 (Ambion) 0.2ul
1mM ATP (or mix) 0.25ul
0.5U/ul PAP (1:10d in H2O) 0.25ul
dH20 0.58ul
Total 4ul


Incubate @ 37C 10 min