Von Ahlfen et al., 2007 [1] described the different factors (e.g. fixation, storage time and conditions) that can influence the integrity of RNA extracted from FFPE tissues, and its downstream applications. They showed that differences in storage time and temperature had a large effect on the degree of RNA degradation. In their study, RNA samples extracted within 1 to 3 days after formalin fixation and paraffin embedding maintained their integrity. Similarly, RNA isolated from FFPE samples that were stored at 4°C showed higher quality compared to samples stored at room temperature or at 37°C. They also reported that RNA fragmentation occurs gradually over time. It is also known that cDNA synthesis from FFPE-derived RNA is limited due to the use of formaldehyde during fixation. Formaldehyde induces chemical modification of RNA, characterized by the formation of methylene crosslinks between nucleic acids and protein. These chemical modifications can be partially irreversible [2], limiting the application of techniques such as reverse transcription, which uses mRNA as a template for cDNA synthesis. A fixation time over 24 hours was shown to result in a higher number of irreversible crosslinks [3,4]. Overall, fixation time and method of RNA extraction are the main factors that determine the extent of methylene crosslinks [1].

A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA expression quantification using low amounts of total RNA [5]. This technique is based on direct measurement of transcript abundance, by using multiplexed, color-coded probe pairs, and is able to detect as little as 0.5 fM of mRNA transcripts; described in detail in Geiss et al., 2008 [5]. In brief, unique pairs of a capture and a reporter probe are synthesized for each gene of interest, allowing ~800 genes to be multiplexed, and their mRNA transcript levels measured, in a single experiment, for each sample. In addition, in a recent study, mRNA expression levels obtained using NanoString were more sensitive than microarrays and yielded similar sensitivity when compared to two quantitative real-time PCR techniques: TaqMan-based RQ-PCR and SYBR Green I fluorescent dye-based RQ-PCR [5]. Although NanoString and RQ-PCR were shown to produce comparable data in good quality samples, NanoString is hybridization-based, and does not require reverse transcription of mRNA and subsequent cDNA amplification. This feature of NanoString technology offers advantages over PCR-based methods, including the absence of amplification bias, which may be higher when using fragmented RNA isolated from FFPE specimens. In addition, NanoString assays do not require the use of control samples, since absolute transcript abundance is determined for each single sample and normalized against the expression of housekeeping genes in that same sample [5].

A recently published study [13] evaluated the performance of quantitative real-time PCR using TaqMan assays (TaqMan Low Density Arrays platform), for gene expression analysis using paired fresh-frozen and FFPE breast cancer samples. The investigators found a good overall correlation coefficient of 0.81 between fresh-frozen and FFPE samples; however, when they compared individual sample pairs, they found a low correlation of 0.33, with variability of 0.005-0.81. These authors suggested that the extensive RNA sample degradation in FFPE samples is likely the cause for the low correlation coefficients observed across sample pairs [13]. Indeed, Bioanalyzer results for our samples showed that fresh-frozen tissues had a good quality RIN and were suitable for gene expression analysis, while FFPE tissues were degraded and had a low RNA integrity number. This RNA degradation in FFPE samples also resulted in higher Ct values initially detectable by RQ-PCR, with loss of amplifiable templates. The low RIN characteristic of FFPE samples did not seem to have an effect on the efficiency of NanoString results, however, when we compared quantification values obtained using RNA isolated from fresh-frozen vs. FFPE tissues.