Evaluation of Non-Formalin Tissue Fixation for Molecular Profiling Studies

Abstract

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

Comparison of Snap Freezing versus Ethanol Fixation for Gene Expression Profiling of Tissue Specimens

“…In a previous study on the effect of fixatives on the preservation, we also assessed the use of alternative embedding procedures, such as low-temperature melting polyester wax. 1 Higher and more intense RNA smears were seen when embedding was performed with low-temperature melting polyester waxes ( Figure 6A from reference 1 ). This data suggests that in addition to fixation, the conventional paraffin-embedding step, used in most of the laboratories, also significantly influences RNA stability.…”

“…We have recently demonstrated the utility of an alternative fixation method, 70% ethanol followed by paraffin embedding. 1 The advantages of formalin-fixed samples are maintained, including excellent preservation of visual details, while allowing for the recovery and analysis of biomolecules. [1][2][3] For example, RT-PCR for specific genes can be performed on RNA from ethanol-paraffin tissues, including small samples such as microdissected cells.…”

“…[1][2][3] For example, RT-PCR for specific genes can be performed on RNA from ethanol-paraffin tissues, including small samples such as microdissected cells. 1 However, an in-depth evaluation of the global integrity of mRNA in ethanol-paraffin samples has not been performed.…”

“…Ethanol fixation and paraffin embedding followed previously established protocols. 1 Specimens obtained from patients on an IRBapproved protocol at the Catholic University in Santiago, Chile, were snap-frozen in OCT and stored at Ϫ80°C. Three frozen cases (Fr1, Fr2, and Fr3) and three ethanolfixed, paraffin-embedded cases (EP1, EP2, and EP3) were selected for microdissection to obtain matched benign prostatic hyperplasia (BPH) and prostate carcinoma cells.…”

Dual RNA-seq of pathogen and host

“…First, fixation should not affect RNA integrity/quality as a prerequisite for downstream analysis by RNA-seq and second, it should not interfere with GFP fluorescence as this parameter is the basis for FACS-based discrimination of host populations. Here, seven commonly used fixation methods were tested with regard to these two parameters ( Fig (Gillespie et al, 2002), the addition of 'stop solution' (i.e. 95% EtOH/5% phenol) (Eriksson et al, 2003), using the (NH4)2SO4-based reagent RNAlater (Qiagen), or different concentrations of paraformaldehyde (PFA) with or without 2% sucrose (W.-D. Hardt, personal communication).…”