mRNA transcript quantification in archival samples using multiplexed, color-coded probes.
Author(s): Reis PP, Waldron L, Goswami RS, Xu W, Xuan Y, Perez-Ordonez B, Gullane P, Irish J, Jurisica I, Kamel-Reid S
Publication: BMC Biotechnol, 2011, Vol. 11, Page 46
PubMed ID: 21549012 PubMed Review Paper? No
Purpose of Paper
This paper compared RNA quality and the correlation of expression of 20 mRNA transcripts in paired archival frozen and formalin-fixed paraffin-embedded (FFPE) oral carcinoma specimens using NanoString and SYBR Green-based real-time PCR.
Conclusion of Paper
As expected, FFPE specimens had lower mean RNA integrity numbers (RIN) and more modest correlations in mRNA levels measured by NanoString and real-time PCR. The correlation in expression between FFPE and frozen specimens was very strong when levels were determined by NanoString (r=0.90, P<0.0001) but only modest when determined by real-time PCR (r=0.50, P<0.0001). Importantly, the mean correlation between frozen and FFPE samples of individual specimens was also very strong when determined by NanoString (r=0.94) and modest (r=0.54) when determined by real-time PCR.
Studies
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Study Purpose
This study compared RNA quality and the correlation of expression of 20 mRNA transcripts in paired archival frozen and FFPE oral carcinoma specimens using NanoString and SYBR Green-based real-time PCR. Oral carcinoma specimens from 19 patients were divided and either snap-frozen and stored in liquid nitrogen or processed by unspecified methods to produce FFPE blocks. RNA was isolated from frozen specimens using Trizol, further purified using the RNeasy kit, and DNAse treated before use. RNA was isolated from five 10 µm FFPE sections using the RecoverAll Total Nucleic Acid Isolation Kit. RNA quality was assessed by bioanalyzer. For NanoString, 100 ng of RNA was used from each frozen specimen and 400 ng was used from the FFPE specimen but the input used for SYBR Green assays was not specified. The 20 mRNA were selected based on previous reports of over-expression in oral cancer and quantified using both methods.
Summary of Findings:
As expected, FFPE specimens had lower mean RNA integrity numbers (RIN) than frozen specimens (average of 2.3 versus 8.3) and correlations in mRNA levels between NanoString and real-time PCR (0.59 versus 0.78, respectively) were more modest. The expression of mRNA measured by NanoString was strongly correlated between FFPE and frozen specimens (r=0.90, P<0.0001) and samples clustered by specimen rather than preservation method. Further, when investigated by specimen pair, the average correlation in expression by NanoString between frozen and FFPE specimens was 0.94 with a minimum of 0.77 and a maximum of 0.99. In contrast, the correlation between FFPE and frozen specimens when expression was measured by real-time PCR was only modest (r=0.50, P<0.0001) with a similarly modest average correlation observed for individual sample pairs (r=0.54, range 0.19-0.99).
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA DNA microarray RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qRT-PCR Specific Technology platform NanoString
Biospecimen Preservation Type of fixation/preservation Snap frozen
Formalin (buffered)