Biomedical Engineering Reference
In-Depth Information
10.1.3
Methods
10.1.3.1
Stabilization of RNA-Protein Complexes Using UV Cross-Linking
1. Grow the cells in the appropriate culture medium on a 150 mm plate to approxi-
mately 80% confluence. If desired (e.g., see Note 1 of Sect.
10.1.4
), add 100 m M
4-thio uridine to the media and let the cells grow overnight.
2. Wash the cells with cold PBS.
3. Remove the PBS, leaving the cells attached to the plate.
4. Irradiate the cells with 0.15 J/cm
2
of 254 nm UV light using a Stratalinker 2400
system or equivalent. If the cells were grown in the presence of 4-thio uridine,
use 0.15 J/cm
2
of a longer wavelength (365 nm) UV light.
10.1.3.2
Stabilization of RNA-Protein Complexes Using Formaldehyde
1. Remove cells from the culture plate, wash with PBS, and resuspend in 10 mL of
cold PBS. Add formaldehyde to a final concentration of 1%.
2. Incubate at room temperature with slow mixing for 10 min.
3. To quench the reaction, add glycine to a final concentration of 0.25 M and incu-
bate at room temperature for 5 min.
4. Harvest the cells by centrifugation at 250 ×
g
for 4 min at 4 °C.
5. Wash cell pellet twice in ice-cold PBS.
6. Cell pellet can be used immediately or stored at −80 °C for future use.
10.1.4
Notes
1. UV cross-linking of unmodi fi ed RNA to proteins is highly speci fi c but notori-
ously inefficient due to the short-lived nature of the free radical intermediate
formed by irradiation with short wavelength UV light. The inclusion of 4-thio
uridine can dramatically increase the efficiency of cross-linking (Hafner et al.
2010
) .
2. Formaldehyde concentration and incubation time can be adjusted to achieve
maximum cross-linking while minimizing noise due to over-cross-linking. In our
experience, formaldehyde concentrations and the time of incubation are the two
variables that should be optimized empirically. Formaldehyde concentrations are
generally varied from 0.3 to 1.0%. The range of fixation times is usually between
5 min and 1 h.
3. Additional approaches using bifunctional cross-linking reagents and chemical
inducers of dimerization (CIDs) (Harvey et al.
2002
) exist to stabilize RNA-
protein interactions but are not commonly used. Many bifunctional cross-linking
reagents available favor protein-protein cross-linking and thus are not suitable for
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