A vast collection of formalin-fixed and paraffin-embedded (FFPE) tissue samples are currently archived in anatomical pathology laboratories and tissue banks around the world. These samples are an extremely valuable source for molecular biology studies, since they have been annotated with varied information on disease states and patient follow-up, such as disease progression in cancer and prognosis/survival data. Although FFPE samples provide an ample source for genetic studies, formalin fixation is known to affect the quality of DNA and RNA extracted from FFPE samples and its downstream applications, such as amplification by the Polymerase Chain Reaction (PCR) or microarrays [1
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
]. 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
Although NanoString technology has been optimized for gene expression analysis using formalin-fixed samples, to our knowledge we are the first to report the use of this technology for mRNA transcript quantification using clinical, archival, FFPE cancer tissues. In our pilot study, we used the NanoString nCounter™ assay for gene expression analysis of archival oral carcinoma samples. In order to show that mRNA levels obtained by NanoString analysis of FFPE tissues were accurate, we compared quantification data obtained using RNA isolated from paired fresh-frozen and FFPE oral cancer samples. Our goal was to determine whether this technology could be applied for accurate gene expression quantification using archived, FFPE oral cancer tissues. We also aimed to compare whether quantification data obtained by NanoString achieved a higher correlation than data obtained by SYBR Green I fluorescent dye-based RQ-PCR, using the same paired fresh-frozen and FFPE samples.