Berglund lab SHAPE protocol (Devika Gates) (5/23/2011)
Please note that SHAPE needs to be optimized for YOUR RNA. It took me about 6 months of optimization to get this protocol to work for my RNA. Make sure you play with the amount of RNA you initially add, the amount of NMIA you add and the amount of kinased reverse primer you add for your reverse transcription. Also, play around with different RT times and between SuperScript II and III. Depending on how structured or unstructured your RNA is, one may work better than the other.
Also, make sure you look at other published protocols and see how they performed SHAPE. I had to read a lot of SHAPE papers and mix and match different protocols to get something that worked for my RNA.
1. In a 1.5mL eppendorf tube add:
6uL 10uM Rev Primer
3uL P32 ATP
2uL 10X PNK Buffer
2uL T4 PNK
2. Incubate at 37C for 30 min
8. Get a spin column and put 800uL of P2beads. Spin at 3000 rpm (1000rcf) for 1 minute. Add 500uL of HE (HE buffer = 10mM HEPES pH=8 and 1mM EDTA pH=8)
buffer, short spin at 1000rcf. Add another 500uL of HE buffer, do another short spin. Centrifuge for 1 min at 1000rcf. Add 20uL of kinased primer to spin column, centrifuge for 5 min at 1000 rcf.
9. Check concentration on whatman paper using the scintillator.
Buffers for SHAPE
3.3X RNA folding mix
333mM HEPES pH 8.0
100% ETOH 200mLs
3M NaOAC pH 5.2 60mLs
10mM HEPES pH=8
1mM EDTA pH=8
2X denaturing dye-
500uLs 2% bromophenol blue
1mL of 1% Xylene cyanol
1mL 5X TBE (makes 5mLs)
1. To 2ug of RNA add up to a total of 75.3uL of HE buffer and .6uL of .125mg heparin
2. Heat the RNA to 95C for 2 minutes then place the RNA on ice for 2 minutes.
3. Add 32.7uL of 3.3X RNA folding mix. Mix gently by pipetting up and down.
4. Remove the tubes from ice and incubate at 37C for 20 minutes.
5. After incubation do a quick spin and remove 9uLs from tube and aliquot into PCR tubes (use one tube for every NMIA concentration you would like to try and one tube for a negative control). The reason I use PCR tubes is because I incubate my tubes in a thermocycler, you can use and eppendorf tube if you would like. The concentrations I have made up 24nM NMIA 48nM NMIA, 72nM NMIA, 96nM NMIA, and 120nM NMIA stocks. (When you make NMIA dilutions make sure they are in neat DMSO) Also, make sure you aliquot RNA into your sequencing lane tubes (for which you don’t add NMIA or DMSO). I use the 120nM stock which makes the final concentration of NMIA at this point 12nM.
6. Make up different stock concentrations of NMIA and add 1uL of NMIA to the +NMIA tubes. To the negative control add 1uL of neat DMSO.
7. Incubate the reactions at 37C for 45 minutes.
8. Add 20uL of inactivation buffer and 1uL of 20mg/mL glycogen to each tube.
9. Incubate at -20C for 15 minutes and centrifuge at max speed for 30 minutes.
10. Remove ethanol supernatant and redissolve RNA in a total of 11uL of HE buffer (This includes the volume of the kinased, for example I would redissolve the RNA in 8uL of HE buffer and add 3uL of kinased primer for a total volume of 11uL). You can store in -20C or continue.
11. Add a final concentration of 18nM reverse kinased primer
12. Add 1uL of 10mM dNTP mix (add only .5uL of 10mM dNTP mix in sequencing tubes).
13. Incubate at 65C for 5 minutes and 35C for 5 minutes. Place on ice for 1 minute.
14. Add 1.5uL of 10mM ddNTPs in sequencing tubes
15. Quick Centrifuge and add 4uL of 5X first strand buffer, 1uL of .1M DTT, and 1uL of SSIII to each tube.
16. Incubate at 52C for 1 hour
17. Heat at 70C for 15 minutes to inactivate
18. Aliquot into 1.5mL tubes and add 300uL of ddH20 and 300uL of phenol chloroform. Vortex for 5 minutes and spin at 4C at max speed for 5 minutes
19. Suck off top aqueous layer (~250uL) and add 625uL of ETOH and 2uL of glycogen to it.
20. Let sit at -20C for 15 minutes and spin at 4C at max speed for 15 minutes
21. Resuspend pellet in 20uL of HE buffer
22. Add 20uL of 2X denaturing dye
23. Heat sample to 95C for 2 minutes.
24. an electrolyte gradient where the top buffer chamber contains .5X TBE and the bottom buffer chamber contains 2 volumes of 1X TBE and 1 volume of 3M sodium acetate pH 7.0. Do not pre-run gels. Make sure you blow out the wells before loading your sample
25. Load 3uL on a long 8% denaturing sequencing gel or store in -20C
26. Run at 65W for as long as required for your RNA.
27. Take gel apart, put saran wrap on one side of the gel and whatman paper on the other side. Make sure there are no air bubbles.
28. Expose the saran wrap side to a phospho-imaging screen overnight.
In an electrolyte gradient gel, when the bromophenol blue dye nears the bottom of the gel, migration ceases. Please refer to the molecular cloning manual chapter 12 protocols 10-11 for more information of how to run electrolyte gradient gels. And how to insert a shark tooth’s comb and for how long to run your gel.