mRNA transcript quantification in archival samples using multiplexed, color-coded probes

Abstract

BackgroundA recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples.ResultsWe measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18) in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008) by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR). We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. We found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r = 0.90) compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r = 0.50). In addition, NanoString data showed a higher mean correlation (r = 0.94) between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r = 0.53).ConclusionsBased on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.

Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation

“…It utilizes a new technique for the expression profiling. The technical features of the nCounter platform 20,21 made this study feasible, because this system works with small amounts of RNA extracted from formalin-fixed paraffin tissue. Extraction of tissue from paraffin blocks matched to H&E slides allowed accurate sampling of areas of interest based on histomorphologic features.…”

“…10,12 In this study, we chose a different approach by using a new analysis platform, the nCounter system, to assess the expression of several hundred genes. 20,21 This platform yields good results with small quantities of RNA isolated from formalin fixed paraffin embedded tissue without a prior amplification step. This allowed us to look for differences in gene expression in matched samples of plexiform neurofibroma and malignant peripheral nerve sheath tumor isolated from within the same surgical specimens.…”

Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues

“…Previous work has established that new approaches for RNA expression profiling in FFPE correlate well with corresponding fresh-frozen samples. 10,11,32 In this study, we sought to determine the reliability of two approaches for mRNA expression profiling in archival FFPE samples from two representative research cohorts. We selected samples with block ages most typically used for biomarker discovery studies in prostate and ovarian cancer.…”

“…11 As an initial step to developing prognostic and predictive mRNA signatures from archival tumor specimens, we performed head-to-head comparisons of gene expression profiles from prostate and ovarian cancer FFPE specimens from large-scale epidemiologic studies and clinical trials representative of a wide variety of fixation times, block ages, and block storage conditions using two platforms: the NuGen WT-Ovation FFPE System V2 þ Affymetrix GeneChip Human Gene 1.0 ST Array [NuGen (NuGen, Inc., San Carlos, CA) þ Affymetrix (Affymetrix, Santa Clara, CA)] and the NanoString nCounter Cancer panel [NanoString (NanoString Technologies, Seattle, WA)].…”

Reproducible, Quantitative, and Flexible Molecular Subtyping of Clinical DLBCL Samples Using the NanoString nCounter System

“…The desire to develop an approach that provides a flexible, robust, and fully quantitative method for DLBCL segmentation led us to evaluate the recently described NanoString system for gene expression (14)(15)(16). NanoString technology uses digital, color-coded barcodes (codesets) that are attached to sequence specific probes, allowing for fully quantitative and direct measurement of mRNA without amplification.…”