Background: Gene expression profiling can improve the diagnostic ability of kidney allograft injury. Fresh tissue is the favored study material for transcriptomic analysis due to the degradative nature of formalin fixation on nucleic acids in FFPE tissues, but is not always available. In this study, we assess the performance of quantifying mRNAs associated with acute rejection (AR) by barcoded oligos and qPCR on FFPE blocks and tissue submerged in RNA stabilizing solution.

Methods: Kidney tissue from 8 stable and 2 patients with AR were preserved in matched FFPE blocks and RNA stabilizing solution (n = 20). The expression of 19 genes was assayed using the NanoString nCounter system (barcoded oligo) and Fluidigm BioMark (conventional qPCR). Expression of the common rejection module (CRM) gene set in FFPE tissue from independent cohort of 10 patients (5 stable; 5 AR) was quantified by both platforms.

Results: On the nCounter platform, the mean correlation between FFPE and unfrozen tissues across the 19 genes was 0.80 with 95% CI (0.72, 0.89). Similarly, the mean correlation between FFPE and unfrozen was 0.81 with 95% CI (0.67, 0.94) on the Fluidigm platform. The average gene expression correlation between nCounter and Fluidigm on FFPE and fresh tissue was 0.66 with 95% CI (0.53, 0.79) and 0.80 with 95% CI (0.77, 0.84), respectively. The CRM score as calculated by the geometric mean expression of the 11 CRM genes has been previously shown to differentiate stable and AR grafts with high sensitivity and specificity. The average CRM score of AR and stable samples calculated from Fluidigm expression was 9.96 and 1.64, respectively (p=1.3E-3). The CRM scores calculated from the nCounter expression data for AR and stable samples were 7.28 and 1.04, respectively (p=2.5E-04).Conclusion: FFPE is a viable source of genomic analysis with modern RNA extraction techniques and quantification methods. Nanostring and Fluidigm have equivalent performance specs for quantifying gene expression from FFPE and fresh tissue and are highly correlated.

CITATION INFORMATION: Sigdel T, Nguyen M, Dobi D, Liberto J, Hsieh S.-C, Vincenti F, Sarwal M, Laszik Z. Performance Comparison of Barcoded Oligos and Standard qPCR on Matched FFPE and RNAlater-Submerged Human Kidney Allograft Samples. Am J Transplant. 2017;17 (suppl 3).

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