Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Multi-platform analysis of microRNA expression measurements in RNA from fresh frozen and FFPE tissues.

Author(s): Kolbert CP, Feddersen RM, Rakhshan F, Grill DE, Simon G, Middha S, Jang JS, Simon V, Schultz DA, Zschunke M, Lingle W, Carr JM, Thompson EA, Oberg AL, Eckloff BW, Wieben ED, Li P, Yang P, Jen J

Publication: PLoS One, 2013, Vol. 8, Page e52517

PubMed ID: 23382819 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine effect of technology platform on the correlation in miRNA expression between FFPE and frozen specimens. FFPE blocks were stored at room temperature for 2 years. While the authors describe both normal and lung carcinoma specimens, only a single specimen pair was used for their experiments. RNA was extracted in duplicate from the frozen specimen using miRNeasy and from the FFPE specimen using RecoverAll.

   Summary of Findings:

   Very strong correlations in miRNA expression were observed for replicate frozen and FFPE specimens using Affymetrix microarrays (r²=0.971 and r²=0.975, respectively), Agilent microarrays (r²=0.988 and r²=0.984, respectively), Illumina microarrays (r²=0.996 and r²=0.976, respectively), NanoString counting (r²=0.967 and r²=0.951, respectively) and NGS (r²=0.971 and r²=0.954, respectively). The correlations in miRNA expression between matched FFPE and frozen specimens were strong using Agilent microarrays (r²=0.826), NanoString counting (r²=0.890), and HiSeq NGS (r²=0.8683) and very strong using Affymetrix microarrays (r²=0.908), Illumina microarrays (r²=0.937), and Gall NGS (r²=0.9056). 484 miRNA were measured using all platforms, with the highest rates of detection observed using NGS (60-80%) and Illumina microarrays (60%) and only 30-45% of transcripts identified using Affymetrix microarrays, Agilent microarrays and NanoString counting. While each platform ranked miRNA expression differently, the signal intensities were comparable when assessed by Affymetrix microarrays, Agilent microarrays, NanoString counting and NGS, but Illumina microarrays had a much higher maximal signal. When expression of 41 miRNAs in the frozen specimen was normalized to the H1299 cell line, the relative miRNA expression by real-time qRT-PCR was strongly correlated with that by NGS (r²=0.7045) and modestly correlated with that by Affymetrix microarrays (r²=0.6308), Agilent microarrays (r²=0.4937), Illumina microarrays (r²=0.5113) and NanoString counting (r²=0.5932). Similarly, when expression of the FFPE specimen was normalized to the H1299 cell line, the relative miRNA expression of the 37 miRNAs assessed by real-time PCR was modestly correlated with that by Affymetrix microarrays (r²=0.4611), NanoString counting (r²=0.4808) and NGS (r²=0.4720) and weakly correlated by Agilent microarrays (r²=0.3516), and Illumina microarrays (r²=0.3350).

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	Real-time qRT-PCR
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Technology platform	Affymetrix microarrays Agilent microarrays Illumina microarrays NanoString counting NGS
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen

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