Top : Molecular Biology : DNA-Protein Interactions : Footprinting : DMS Chemical Footprinting of RNA

DMS Chemical Footprinting of RNA

Source: Laederach Lab
Date Added: Thu Dec 17 2009
Date Modified: Fri Dec 18 2009
Abstract: Dimethyl Sulfate is used as a chemical probe by methylation of A's and C's. Primer extension with a cy'5 labeled primer and reverse transcription allow us to determine secondary structures of RNA.

Notes:

  1. RNA should be at 1 uM  concentration (1 pmole/uL)
  2. There are 3 conditions (use a blank/background reaction for each condition).

Solutions

CE Buffer (10X)

  • 1M Na Cacodylate (4.28 g into 20mL H2O) pH 7.4
  • 5 mL of 1 M Na Cacodylate
  • 100 uL of 0.5 M EDTA
  • 44.9 mL H2O
    Sterile filter and allow vacuum to continue 5 mins. to allow for degassing.

1X CE Buffer

  • 1 mL 10X CE
  • 9 mL H2O

DMS Solution:

  • 15 uL 100% ETOH
  • 3 uL DMS

Quench Solution:

  • 1.4 mL 2-beta-mercaptoethanol
  • 0.5 mL 3 M Sodium Acetate (Ambion Buffer kit)
  • 3.1 mL H2O

*Dilute RNA to 1 uM in 1x CE buffer

Conditions

Condition 1:

10 uL RNA (1 uM concentration)
2.5 uL 10x CE buffer
12.5 uL H2O

Condition 2:

10 uL RNA (1 uM concentration)
2.5 uL 10x CE buffer
1.25 uL 2 M KCl (Ambion Buffer kit)
11.25uL H2O

Condition 3:

10 uL RNA (1 uM concentration)
2.5 uL 10x CE buffer
1.25 uL 2 M KCl (Ambion Buffer kit)
*1.25 uL 100 mM MgCl2 (diluted from 1M MgCl2 Ambion Buffer kit)
10 uL H2O
*DO NOT ADD MgCl2 UNTIL READY TO INITIATE FOLDING.

Procedures

Folding Steps

  1. Heat the 25 ul (Mg free) reaction mix at 90C for 3 mins.
  2. Take the sample out and let it cool to room temperature for about 10-15 mins-, after which give a quick spin on a table top centrifuge.
  3. Put the sample in a 50C heating block and let it equilibriate at 50C for 10 mins.
  4. Add MgCl2 (final Mg concentration 10 mM) to samples.
  5. Place sample in a 50C heating block for 15-30 mins.
  6. Take the sample out and put it in a heating block set at the folding temperature (25C or 37C). Incubate at the folding temperature for 1 hour.
  7. Initiate modification reaction (OH footprinting/DMS....etc.) at the same temperature used for folding (25C or 37C).
  8. Similarly, we prepare the unfolded sample in an identical way but skip the addition of MgCl2 at step 3.

DMS modification

  1. To the 12.5 uL of folded RNA, add 0.5 uL of DMS solution
  2. Incubate for 2 minutes at 25C or 37C.
  3. Add 475 uL of quench solution (even to samples without DMS mod)
  4. Add 1 mL of 100% ETOH
  5. Freeze O/N at -80C
  6. Prepare RNA for RT

Reverse Transcription

  1. Spin down samples at 14,000 RPM at 4C for 1 hour
  2. Remove supernatant
  3. Rinse with 100 uL of 70% ETOH
  4. Spin at 14,000 RPM at 4C for 0.5 hours
  5. Remove supernatant and dry pellet in speed-vac, medium heat for 3 mins.

RT Buffers

Annealing Buffer (50 mM Tris-CL, pH 8.3, 60 mM NaCl, and 10 mM DTT)

Tris-Cl    5 uL
NaCl    1.1 uL
DTT    10 uL
H2O    83.9 uL
Total volume  100 uL

Reverse Transcription Mix

5x FS buffer      4 uL per rx
0.1 mM DTT      1 uL per rx
RNase Out      2 uL per rx
10 mM dNTP mix      2 uL per rx
*ddNTP 5 mM      5 uL per rx
Superscript III enzyme    1 uL per rx

* Only use if doing a dideoxy sequencing reaction.

RT Reaction

  1. Resuspend RNA pellet in 9 uL of annealing buffer
  2. Add 1 uL of cy5 labeled primer at 10 uM
  3. Heat to 90C for 2 min. and slowly cool to 25C to allow primer to anneal. (1-1.5 hours)
    Note: Keep tubes in heat block and pull out whole block from heat. Use a thermometer to monitor the temp.
  4. Add 9 uL of reverse transcriptase mix
  5. Heat solution at 55C for 5 mins
  6. THEN add 1 uL of superscript III enzyme to each sample.
  7. Mix gently, briefly centrifuge, incubate at 55C for 10-15 mins.

Clean Up Sample

  1. Add 2 uL of 2N NaOH
  2. Incubate at 95C for 3 mins.
  3. Add 2 uL of 2N HCl to neutralize
  4. Add 3 uL of 3 M Na-acetate
  5. Add 1 uL of 100 mM MgCl2
  6. Add 90 uL of 100% ETOH
  7. Centrifuge at 14,000 RPM for 30 mins at 4C
  8. Remove supernatant
  9. Add 50 uL 70% ETOH
  10. Centrifuge at 14,000 RPM for 30 mins at 4C
  11. Dry pellet and re-suspend in 60 uL Sample Loading Solution (Beckman)
  12. Load 60 uL of sample in 96 well plate
  13. Add 1 uL of Ladder to each well
  14. Place drop of mineral oil on top and follow CEQ protocol

References:

  1. Mitra, S, Brenowitz, M, Laederach, A, Altman, R., Semi-automated capillary separation footprinting analysis. 2007.
Printer friendly page