• Process previously frozen tissues like freshly harvested samples
  • Thawing tissues in RNAlater®-ICE protects RNA from degradation
  • No more tissue pulverizing with mortar and pestle and awkward transfer of powder to tube
  • Easily apportion frozen tissue samples for multiple experiments

RNAlater-ICE Frozen Tissue Transition Solution is a unique RNA stabilizing solution. Simply drop frozen tissues into RNAlater-ICE and walk away! Once tissues are thawed they can be easily processed using standard RNA isolation procedures. No more laborious grinding of frozen tissue to preserve RNA in difficult tissues or tissues that need to be stored prior to isolation. Treated tissues can be directly inserted into standard homogenization and isolation protocols and processed as though fresh.

Quick Freezing Tissues Preserves RNA

Historically, tissues that need to be stored prior to RNA isolation are "snap" or "flash" frozen on dry ice or in liquid nitrogen to preserve RNA integrity. RNA in tissue is stable while frozen at -80ºC but thawing the tissue prior to or during its disruption can result in RNA degradation. This is true even if the tissue thaws while in the denaturation solution.

Processing Frozen Tissues is Problematic

Frozen tissues are typically ground with a chilled mortar and pestle for quality RNA isolation. Liquid nitrogen must be added to the mortar to keep the sample frozen while it is ground. For multiple samples this process is laborious. Either a separate mortar and pestle set is needed for each sample, or the set must be thawed and cleaned after each sample is processed to avoid cross-contamination. Powdered tissue can also thaw during transfer to a homogenization vessel. This often results in formation of clumps that do not readily disperse in the lysis solution, resulting in RNA degradation and loss. Very small samples should be homogenized immediately in lysis solution, which can again be cumbersome if there are multiple samples to process.

Process Frozen Tissue Without Jeopardizing RNA Integrity — RNAlater-ICE

RNAlater-ICE solves all of these problems. Simply submerge frozen tissue samples in 10 volumes of RNAlater-ICE and store overnight at -20 or -80ºC (the solution will remain liquid at these temperatures). As the tissue thaws, RNA integrity is protected. Once treated, tissue can be safely stored at 4ºC or even at room temperature (for a limited period of time) and can be further dissected or processed prior to homogenization in a standard RNA isolation lysis buffer. Thus the same frozen tissue sample can be used multiple times for different experiments without compromising RNA integrity.

Figure 1 shows the quality of RNA isolated from three different frozen mouse tissues that were immediately homogenized or thawed in RNAlater-ICE overnight at -20ºC. Both the stained gel and the resulting Northern blot demonstrate that RNAs isolated from tissues treated with RNAlater-ICE maintain a high degree of integrity. The dramatic preservation of RNA by RNAlater-ICE is also illustrated in Figure 2. Here we compare RNA that was prepared directly by homogenization of frozen tissue, with a sample that was allowed to thaw at room temperature for 5 minutes, and a sample that was frozen, soaked overnight at -20ºC in RNAlater-ICE, and kept at room temperature for 30 minutes before being processed for RNA isolation. The protective effect of RNAlater-ICE is obvious.

Figure 1. Quality of RNA From Samples Treated With RNAlater-ICE. Total RNA was isolated from various frozen mouse tissue samples that were either processed directly from a frozen state or thawed in RNAlater-ICE at -20ºC overnight. (A) shows the ethidium bromide stained RNA in a denaturing agarose gel. (B) shows the results of Northern blot analysis of the same gel hybridized to radiolabeled probes for ß-actin, GAPDH, and cyclophilin. (Note that the specific activity of the cyclophilin probe was lower than that of the other probes.) For each tissue the integrity of both rRNA and mRNAs for the RNAlater ICE treated samples is comparable to that of RNA prepared directly from frozen tissue samples.

Figure 2. RNA Integrity from Frozen Samples Ground, Thawed or Treated with RNAlater-ICE. Total RNA was isolated from mouse liver samples that were processed directly from a frozen state (ground), thawed on a benchtop for 5 min (thawed), or thawed overnight at -20ºC in RNAlater-ICE and then stored at rt for 30 min prior to RNA isolation (treated). (A) shows the ethidium bromide stained RNA in a denaturing agarose gel. (B) shows the results of Northern blot analysis of the same gel hybridized to radiolabeled probes for ß-actin, GAPDH, and cyclophilin. Note that frozen tissue thawed in the absence of RNAlater-ICE yielded degraded RNA while RNA remained intact when tissue was thawed in RNAlater-ICE.