Research Paper Volume 5, Issue 2 pp 120—129

Figure 3. Effects of formaldehyde crosslinking, crosslink reversal, and permeabilization on RNA extraction and quantification of gene expression by qPCR. (A-C) Effect of crosslink reversal. (A) Cells were harvested by trypsinization, fixed with paraformaldehyde in solution, permeabilized with Triton X-100, and crosslinks were reversed by incubation in a buffer containing 200 mM NaCl and 1% SDS for 2 hr at 65°C. RNA was subsequently purified by phenol extraction, ethanol precipitated, and analyzed on a Bioanalyzer instrument. (B) Cells were processed as in panel A, but the crosslink reversal step was omitted. (C) Equal amounts of RNA preparations (1 μg) from (a) and (b) were reverse transcribed and qPCR was performed with primers to GAPDH and p16 (gene symbol CDKN2A) genes. Control was total RNA that was prepared directly from cells using Trizol reagent. (D-F) Effect of cell premeabilization. (D) Cells were grown on cover slips and processed for immunofluorescent staining of p16 using a protocol that includes permeabilization with Triton X-100 (left panels). The permeabilization step was omitted in the right panels. (E) Cells were processed as in panel (a), except that for lane 1 the permeabilization step was omitted (cells were incubated for an equivalent amount of time in buffer without Triton X-100). Control was total RNA that was prepared directly from cells using Trizol reagent. (F) Equal amounts of RNA preparations (1 μg) from (e), lanes 1-3, were reverse transcribed and qPCR was performed as in panel (c).