Setting out the frame conditions for feasible use of FFPE derived RNA.
Author(s): Schmeller J, Wessolly M, Mairinger E, Borchert S, Hager T, Mairinger T, Schmid KW, Wohlschlaeger J, Walter RFH, Mairinger FD
Publication: Pathol Res Pract, 2019, Vol. 215, Page 381-386
PubMed ID: 30606660 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to identify an optimal storage temperature for slide-mounted formalin-fixed, paraffin-embedded (FFPE) sections slated for RNA analysis and to assess potential effects introduced by different methods of deparaffinization and RNA isolation on qRT-PCR analysis and concentrations of total and long fragment (150-6000 nt) RNA.
Conclusion of Paper
Due to significantly lower RNA concentrations (total and long fragment) and RNA Quality Numbers (RQNs) that were observed at warmer temperatures, the authors advise storing slide-mounted FFPE sections at -20°C or colder. While no clear effects of deparaffinization method were observed, RNA isolation with the Promega Maxwell RSC RNA FFPE Kit produced lower concentrations of total (P=0.0023) and long fragment RNA (P=0.0073) but a higher relative ratio of long fragment to total RNA (P<0.0005), indicating superior quality in comparison to the Qiagen RNeasy Kit. The authors report that Ct values of beta actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), chemokine ligand 18 (CCL18), and proteasome alpha subunits (PSMAS) were not significantly affected by RNA isolation method, although data was not shown.
Studies
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Study Purpose
This aim of this study was to identify the optimal temperature for storing unstained FFPE slide-mounted sections for subsequent analysis of RNA transcripts by qRT-PCR. Retrospectively collected lung tissue specimens were analyzed from three otherwise healthy patients diagnosed with spontaneous pneumatothorax. FFPE blocks were stored for approximately 3 y prior to sectioning. Slides containing 10 µm-thick FFPE sections were stored for 1 week at 24, 4, -20, or -80°C prior to deparaffinization in xylene and RNA isolation with the Qiagen RNeasy Kit. Two (10 µm-thick) sections were analyzed for each sample. RNA quantity was determined using the Qubit fluorometer and RNA integrity was assessed by a capillary electrophoresis-based fragment analyzer.
Summary of Findings:
The storage temperature of slide-mounted FFPE sections significantly affected total RNA concentration (P=0.0175), the abundance of long (150-6000 nt) RNA fragments (P=0.0028), and RNA Quality Number (P=0.0045) with higher values observed at colder temperatures for all (-80°C > -20°C > 4°C > 24°C). The authors report that the relative ratio of short (<150 nt) to long RNA fragments remained stable across storage temperatures, although data was not shown.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Other diagnoses
- Normal
Platform:
Analyte Technology Platform RNA Electrophoresis RNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 24°C
4°C
-20°C
-80°C
-
Study Purpose
The purpose of this study was to identify the superior method of FFPE slide deparaffinization and RNA extraction for analysis of RNA quantity, integrity, and relative gene expression. Retrospectively collected lung tissue specimens were analyzed from twenty otherwise healthy patients diagnosed with spontaneous pneumatothorax. FFPE blocks were stored for 3-4 y prior to sectioning and slide-mounted FFPE sections were stored at -20°C for 1 week prior to deparaffinization with xylene, mineral oil, or d-Limonene. RNA was isolated with either the Promega Maxwell RSC RNA FFPE Kit or the Qiagen RNeasy FFPE Kit. Slides containing 10 µm-thick FFPE sections were stored for 1 week at 24, 4, -20, or -80°C prior to deparaffinization in xylene and RNA isolation with the Qiagen RNeasy Kit. Two (10 µm-thick) sections were analyzed for each sample. RNA quantity was determined using the Qubit fluorometer and RNA integrity was assessed by a capillary electrophoresis-based fragment analyzer. Levels of PSMA5, CCL18, ACTB, and GAPDH transcripts were determined by qRT-PCR. Briefly, RNA was reverse transcribed with random hexamers and stored at -20°C until amplification with a TaqMan Gene Expression assay.
Summary of Findings:
No clear differences were observed among the different deparaffinization methods examined in concentrations of total RNA or long RNA fragments when RNA was isolated using the Qiagen RNeasy kit (xylene versus d-Lim) or the Promega Maxwell RSC RNA FFPE kit (mineral oil versus d-Lim). When different RNA isolation methods were examined, slide-mounted FFPE sections (10 µm-thick) that were deparaffinized with d-Lim had significantly greater concentrations of total RNA (P=0.0023) and long RNA fragments (P=0.0073) when RNA was isolated with the Qiagen RNeasy Kit than the Promega Maxwell RSC RNA FFPE Kit; however, the relative ratio of long RNA fragments to total RNA was greater (indicating superior RNA integrity) when RNA was isolated with the Promega Maxwell RSC RNA FFPE Kit (P<0.0005). The authors report that Ct values of ACTB and GAPDH did not significantly differ as a result of different RNA isolation kits, although data was not shown, and Ct values of the negative controls CCL18 and PSMAS were higher than 35, as expected.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Other diagnoses
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Deparaffinization Xylene
Mineral oil
d-Limonene
Real-time qRT-PCR Specific Targeted nucleic acid PSMA5
CCL18
ACTB
GAPDH
Analyte Extraction and Purification Analyte isolation method Promega Maxwell RSC RNA FFPE Kit
Qiagen RNeasy FFPE Kit