Comparing Quality Between Frozen and FFPE Breast Cancer Samples

Storage of breast cancer tissue samples as formalin fixed paraffin-embedded (FFPE) blocks is a common way to preserve tumor samples long-term. These blocks can be stable for several decades, enabling longitudinal studies critical to advancing our understanding of the disease pathology. Breast cancer is not a single disease; it is a collection of heterogeneous diseases each having distinct molecular subtypes. Microarray analysis has had a profound effect on expanding our understanding of breast cancer and overcoming the barriers presented by the genetic heterogeneity of the disease. FFPE samples exist in every pathology department so an ability to utilize them as sources for molecular-level research would add a wealth of data to the oncologist’s repository of information.

The vast archives of FFPE samples provide a valuable source of RNA, but problems exist in using them for gene expression profiling. The quality of the isolated RNA is low and this poses a major challenge for downstream analyses like PCR and microarray.

Fresh frozen tissue samples lack the longevity of their formalin fixed counterparts but they provide much higher quality RNA. By avoiding the fixation process, frozen tissue derived RNA is subjected to significantly less chemical modifications. Some prevalent fixation induced chemical modifications include: cross linking to proteins, Poly-A tail removal, formaldehyde mediated reactions between nucleic acids, RNA strand fragmentation and 3’ amplification by oligo-dT primers. These modifications are all prevalent problems found in FFPE obtained RNA samples and largely absent in frozen tissue blocks. 

Frozen tissue derived RNA is not without problems though. The main obstacle in maintaining RNA integrity when working with frozen tissue lies in the thawing step. Tissue samples must be thawed sometimes before proceeding to downstream applications and this step can cause RNA degradation. Repetitive use of tissue for analysis amplifies this problem by subjecting the sample to multiple cycles of freezing and thawing. Though RNA degradation is minimal, especially when compared to fixation induced degradation, thawing for more than thirty seconds increases the likelihood that RNA profiles will be affected. 

Fresh frozen samples, regardless of their minor imperfections, remain the gold standard for RNA analysis. Though frozen samples of breast cancer tissue are less abundant than FFPE blocks due to longevity, they provide easily extracted and reliable RNA that will help develop personalized treatment regimens by clarifying the genetic heterogeneity of breast cancer.

It should be noted that obtaining reliable and valuable data from degraded RNA is not impossible. If RNA quality is accurately assessed and identified as compromised, methods can be used to circumvent the problems posed by fixation-induced chemical modifications. Kits for assays that specifically address the problem of FFPE RNA quality are available and show impressive peer-reviewed efficacy. 

These assay kits, by enabling RNA analysis in FFPE blocks, should greatly expand upon our understanding of breast cancer at the molecular level. Longitudinal analysis of expressed RNA transcripts will impart a welcomed new utility on the vast archives of fixed tissue samples that have existed in repositories for decades. 


Biomed Central - RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces

Biomed Central - RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies