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.