RNA Detection

ice-cold PBS. Collect cells and freeze pellets in liquid nitrogen

or continue with lysis (seeNote 19).

7. Lyse ‘light” and “heavy” cells in approximately five times the
   cell pellet volume of lysis/binding buffer and incubate the
   extract for 15 min on ice.


8. Pass the lysate several times through a 23- and 26-G needle.
   Save an aliquot (0.2 mL) for downstream analyses and store it
   at 80 C.


9. Spike-in the same volume of the heavy lysate into untreated and
   treated “light” cell lysates. We typically mix “heavy” to “light”
   lysates in a 1:5 (v/v) ratio (seeNote 20).


10. Prewash the magnetic oligo(dT) beads in 5 mL of lysis/bind-
    ing buffer. Afterward, resuspend them into the cell extract by
    first taking them up in 1 mL of the prepared lysate and trans-
    ferring them to the tube containing the remaining lysate.


11. Perform the incubation, washing, and elution steps as
    described in Subheading3.2.

AB

10

15

20

25

30

50

85

120

200

kDa

input

(0.0015%)

IR - +

eluate

(8%)

-+

protein amount (ug)

0.1 0.25 0.5 1 5

15

20

25

30

50

60

85

120

200

kDa

input (0.02%)eluate (4%)

UV

4SU +

+

+

+

+

+

*

*

nucleases nucleases

Fig. 2Quality control and analysis of oligo(dT) eluates. (a) Isolation of poly(A)+RNA binding proteins. MCF-7
cells were incubated in the presence of 200μM 4SU for 16 h, UV cross-linked (0.2 J/cm^2 ) or left untreated.
After lysis and oligo(dT) purification, mRNA complexes were eluted and RNase treated. Indicated fractions of
total input and eluate volumes were resolved with SDS-PAGE. Proteins were visualized using silver staining.
(b) Example of determination of differential protein binding to poly(A)+RNA upon DNA damage induction. MCF-
7 cells were incubated in the presence of 200μM 4SU for 16 h, followed by ionizing radiation (IR) exposure
(10 Gy) to induce double strand breaks, and UV cross-linking (0.2 J/cm^2 ). After oligo(dT) affinity purification,
protein–mRNA complexes were eluted and RNase treated. Indicated fractions of total input and eluate
volumes were resolved with SDS-PAGE. Proteins were visualized using silver staining. To judge the amount
of proteins in oligo(dT) eluates, increasing amounts (0.1–5μg) of an MCF-7 NP40 lysate with a known
concentration were loaded on the same gel

412 Miha Milek and Markus Landthaler