Standard riboprobe synthesis protocol

1. Assemble reaction at room temperature:
2. 2 μl RNA Polymerase (SP6, T7 or T3; 20U/μl; thus far, we have tried RNA Pol. from Ambion, NEB, and Thermo, and they all work pretty well)
3. 4 μl 5X RNA Pol buffer or 2 μl 10X RNA Pol buffer
4. 1 μl RNase inhibitor
5. 2 μl 10X NTP labeling mix [digoxigenin RNA labeling mix (Roche 11277073910), biotin RNA labeling mix (Roche 11685597910), fluorescein RNA labeling mix (Roche 11685619910), or homemade labeling mix (see below for recipe)]
6. X μl linear template DNA (0.5-1 μg in total)
7. 11-X μl RNase-free water, if use 5X RNA Pol buffer (e.g. Thermo) or 13-X μl RNase-free water, if use 10X RNA Pol buffer (e.g. Ambion, or NEB)
8. (total volume = 20 μl)
9. Mix well, spin down and then incubate at 37°C for several hours (up to overnight).
10. (Optional) Add 1 μl of RNase-free DNase, and incubate at 37°C for 30 mins.
11. Precipitate RNA by EtOH.
12. Add 4 μl 6M lithium chloride to the reaction, mix well and spin down, add 100 μl cold 100% EtOH, mix well, and store at -20°C for at least 1hr (up to overnight). Centrifuge at maximum speed in 4°C for 15 min to pellet the RNA.
13. Note that the pellet should be visible. Wash pellet with cold 75% EtOH, dry the pellet at room temperature for 10 minutes.
14. Dissolve RNA with 12 μl nuclease-free ddH2O. Quantify RNA concentration. Add appropriate amount of PreHyb to bring the final concentration to 100 ng/μl.
15. Or, simply dissolve the RNA pellet in 100 μl PreHyb.
16. Store the probe stock in -20°C.

This protocol regularly yields ~10 μg riboprobe per each 20 μl reaction.

Protocol for hydrolyzed riboprobe synthesis (to produce smaller probe for better penetrance; not essential but may improve the signal/noise ratio for young adults, juveniles or very-late-stage embryos)

1. Assemble reaction at room temperature:
2. 2 μl RNA Polymerase (SP6, T7 or T3; 20U/μl; thus far, we have tried RNA Pol. from Ambion, NEB, and Thermo, and they all work pretty well)
3. 4 μl 5X RNA Pol buffer or 2 μl 10X RNA Pol buffer
4. 1 μl RNase inhibitor
5. 2 μl 10X NTP labeling mix [digoxigenin RNA labeling mix (Roche 11277073910), biotin RNA labeling mix (Roche 11685597910), fluorescein RNA labeling mix (Roche 11685619910), or homemade labeling mix (see below for recipe)]
6. X μl linear template DNA (0.5-1 μg in total)
7. 11-X μl RNase-free water, if use 5X RNA Pol buffer (e.g. Thermo) or 13-X μl RNase-free water, if use 10X RNA Pol buffer (e.g. Ambion, or NEB)
8. (total volume = 20 μl)
9. Mix well, spin down and then incubate at 37°C for several hours (up to overnight).
10. (Optional) Add 1 μl of RNase-free DNase, and incubate at 37°C for 30 mins.
11. Precipitate RNA by EtOH.
12. Add 4 μl 6M lithium chloride to the reaction, mix well and spin down, add 100 μl cold 100% EtOH, mix well, and store at -20°C for at least 1hr (up to overnight). Centrifuge at maximum speed in 4°C for 15 min to pellet the RNA.
13. Note that the pellet should be visible. Wash pellet with cold 75% EtOH, dry the pellet at room temperature for 10 minutes.
14. Dissolve RNA with 40 μl nuclease-free ddH2O.
15. Add 4 μl 0.5 M sodium bicarbonate and 6 μl 0.5 M sodium carbonate (pH would be ~10). Mix.
16. Incubate in 60°C for X min. X= [(original length of the probe, in kb) - (desired length of the probe, in kb)]/[0.11 * (original length of the probe, in kb) * (desired length of the probe, in kb)]
17. Neutralize reaction by adding 200 μl nuclease-free ddH2O, 13.75 μl 3 M NaOAc, 1.25 μl glacial acetic acid, 50 μl 6M lithium chloride. Add 800 μl cold 100% EtOH, mix well, and store at -20°C for at least 1hr (up to overnight). Centrifuge at maximum speed in 4°C for 15 min to pellet the RNA.
18. Wash pellet with cold 75% EtOH, dry the pellet at room temperature for 10 minutes.
19. Dissolve RNA with 13 μl nuclease-free ddH2O. Quantify RNA concentration. Take 1 μl to run gel electrophoresis for monitoring the outcome of probe hydrolysis.
20. Add appropriate amount of PreHyb to bring the final concentration to 100 ng/μl.
21. Store the probe stock in -20°C.

Recipe for 10X labeling mixture

22.5μl ddH2O

7.5μl 100 mM ATP

7.5μl 100 mM CTP

7.5μl 100 mM GTP

5.0μl 100 mM UTP

25.0μl digoxigenin/biotin/fluorescein-UTP (10mM; Roche 11209256910/11388908910/11427857910); DNP-UTP (10mM; PerkinElmer NEL555001EA); Aminoallyl-UTP (diluted into 10mM from 50mM stock; Ambion AM8437 or Thermo R1091; aminoallyl labeling is for subsequent tagging labeled RNA with amine-reactive dye or hapten, not for direct antibody detection)

(total = 75μl)

Recipe for carbonate buffer

0.5 M sodium bicarbonate: 2.1 g sodium carbonate, add ddH2O to a 50 ml total volume.

0.5 M sodium carbonate: 2.65 g sodium carbonate, add ddH2O to a 50 ml total volume.