Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays.
Author(s): Haynes HR, Killick-Cole CL, Hares KM, Redondo J, Kemp KC, Moutasim KA, Faulkner C, Wilkins A, Kurian KM
Publication: J Clin Pathol, 2018, Vol. 71, Page 695-701
PubMed ID: 29463577 PubMed Review Paper? No
Purpose of Paper
This study investigated whether storage of formalin-fixed, paraffin-embedded (FFPE) glioblastoma and temporal lobe tissue blocks for up to 60 months prior to RNA extraction affects RNA yield, purity, or integrity.
Conclusion of Paper
Glioblastoma specimens had a significantly lower mean RNA Integrity Number (RIN) than control temporal lobe specimens collected from epilepsy patients (1.7 versus 2.2, respectively; p=0.008), although the storage duration of FFPE blocks were comparable. No significant correlations were observed between FFPE block storage and RIN, RNA yield, or 260/280 ratio. Ct values for GAPDH and 18S were significantly and negatively correlated to 260/280 ratio in glioblastoma and control epilepsy specimens (p=0.006 and p=0.013, respectively).
Studies
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Study Purpose
The purpose of this study was to assess the suitability of RNA isolated from glioblastoma and temporal lobe specimens stored as FFPE blocks for up to 60 months for gene expression analysis by qRT-PCR. RNA yield, purity, and qRT-PCR suitability was assessed in microdissected areas (unstained) from 48 glioblastoma FFPE specimens and results were compared to whole sections (unstained) of 17 temporal lobe specimens obtained following surgical resection for epilepsy. The authors do not specify the conditions of formalin fixation and paraffin processing. Ten 5 µm-thick sections collected from each specimen were stored at ambient temperature in the dark for no longer than 21 d prior to RNA extraction with the EZRNA FFPE RNA kit from Omega Bio-tek with a gDNA elimination spin column. Isolated RNA was stored at -80°C until analysis for concentration by a Nanodrop spectrophotometer, RNA integrity by a bioanalyzer, and quantification of the reference genes GAPDH and 18S by qRT-PCR. RNA for qRT-PCR analysis was reverse transcribed using a Clontech TaKaRa PrimeScript first strand cDNA Synthesis Kit and data was assessed using the 2-ΔΔCt method.
Summary of Findings:
Expression of the reference genes GAPDH and 18S (when normalized to one another) was not significantly different between glioblastoma and control epilepsy FFPE specimens. Mean RIN was significantly lower for glioblastoma specimens compared to control epilepsy specimens (1.7 versus 2.2; p=0.008), although the storage duration of FFPE blocks was comparable (glioblastoma specimens: 3-60 months, epilepsy specimens: 14-55 months). Glioblastoma and control epilepsy specimens had comparable 260/280 ratios (1.95 versus 1.94, respectively). RNA yield was not correlated to RIN or 260/280 ratio when glioblastoma and epilepsy specimens were combined. The age of the FFPE block at the time of RNA extraction was not correlated to RIN, RNA yield, or 260/280 ratio in control epilepsy or glioblastoma specimens, although a nonsignificant negative trend was observed between RNA yield and block age for glioblastoma specimens. Raw cycle threshold (Ct) values were not correlated to RIN when glioblastoma and control epilepsy specimens were combined, although a non-significant trend was observed for 18S expression analysis. Ct values for GAPDH and 18S and 260/280 ratio was significantly and negatively correlated in glioblastoma and control epilepsy specimens (p=0.006 and p=0.013, respectively).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Epilepsy
- Neoplastic - Mixed type
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 3-60 months
Real-time qRT-PCR Specific Targeted nucleic acid GAPDH
18S
Preaquisition Diagnosis/ patient condition Epilepsy
Glioblastoma