Formalin vs. Frozen Sections
LCM is usually carried out on frozen sections when samples will be used for RNA isolation. This is because formalin fixation introduces crosslinks between nucleic acids and proteins, resulting in fragmentation of the RNA. Freezing avoids crosslinking but introduces a different problem. Endogenous nucleases are not irreversibly inactivated, and can become active during the tissue processing steps (fixation, staining, destaining, and dehydration) carried out after sectioning.
An Improved Protocol for Staining
Ambion scientists performed a detailed analysi s of RNA recovered from LCM sections after various steps of the conventional post-sectioning processing protocols for frozen samples. These studies revealed that significant RNA degradation occurred during exposure of the section to aqueous solutions, presumably due to reactivation of endogenous nucleases. To circumvent this problem, Ambion developed a staining procedure that avoids exposing the sections to water at any step. (See our LCM Staining Kit.) Using the optimized "water-free" tissue processing protocol in conjunction with the RNAqueous-Micro Kit, total RNA of excellent quality, as assessed by microfluidic capillary electrophoresis and real-time RT-PCR, can be obtained from pure populations of cells selected by LCM (Figure 1).
Figure 1. Real-Time RT-PCR of ICM Samples. Mouse spleen (A) and liver (B) samples (1 mg, 3 mg and 10 mg) were dissected by LCM and RNA was isolated using the RNAqueous-Micro Kit according to protocol. The RNA was then used as a template for the amplification of GAPDH by real-time RT-PCR.