Influence of formalin fixation duration on RNA quality and quantity from formalin-fixed paraffin-embedded hepatocellular carcinoma tissues.
Author(s): Amemiya K, Hirotsu Y, Nagakubo Y, Mochizuki H, Oyama T, Omata M
Publication: Pathol Int, 2023, Vol. 73, Page 593-600
PubMed ID: 37933792 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if the duration of formalin fixation (1-240 h) and the duration of formalin-fixed, paraffin-embedded (FFPE) block storage affect the quality and quantity of RNA extracted from a single hepatocellular carcinoma (HCC) specimen or next-generation sequencing (NGS) results. Three methods of quantifying RNA concentration were also compared using one HCC specimen: spectrophotometry (Nanodrop), fluorometry (Qubit), and automated electrophoresis (Bioanalyzer).
Conclusion of Paper
The measured total RNA yield differed among the three quantification methods evaluated (Qubit, NanoDrop, Bioanalyzer); however, the duration of formalin fixation led to a decline in total RNA yield per unit surface area for all three quantification methods, with blocks fixed for 48 h yielding just 24-38% of the RNA obtained from blocks fixed for 2 h, depending on quantification method. The total amount of RNA that was longer than 200 nt (calculated by multiplying the total RNA amount by the percentage of RNA fragments longer than 200 nt (DV200 value)) declined progressively with time in fixative, although RNA integrity numbers (RINs) were not affected. Library concentration (measured prior to equalization) was negatively correlated with the duration of fixation (r= - 0.9181), and tissue blocks that were fixed longer than 72 h yielded libraries below the concentration recommended prior to equalizing (100 pmol/L). Although the duration of formalin fixation was not correlated to the total number of mapped reads (r = - 0.12), it was negatively correlated to the number of mapped reads for each of the following internal control genes: HMBS (r = -0.79), ITGB7 (r= -0.79), and MYC (r= -0.89). When blocks containing FFPE tissue that was fixed for 1-12 h were stored for 500 d at room temperature, reductions were observed in the total amount of RNA (determined by bioanalyzer) per unit tissue area, DV200 values (the percentage of RNA fragments > 200 nts), and the total amount of RNA that was longer than 200 nt relative to when the same tissue was analyzed immediately.
Studies
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Study Purpose
The purpose of this study was to determine if the duration of formalin fixation (1-240 h) and the duration of formalin-fixed, paraffin-embedded (FFPE) block storage affect the quality and quantity of RNA extracted from a single hepatocellular carcinoma (HCC) specimen or next-generation sequencing (NGS) results. Three methods of quantifying RNA concentration were compared: spectrophotometry (Nanodrop), fluorometry (Qubit), and automated electrophoresis (Bioanalyzer). A single hepatocellular carcinoma (HCC) surgical resection specimen was divided into 11 blocks (5 mm2), and each tissue block was fixed in 10% neutral buffered formalin at room temperature for 1, 2, 4, 6, 12, 24, 48, 72, 120, 168, or 240 h. For each time point, 5-10 tissue sections (5 µm-thick) were deparaffinized in xylene, tumor cells were isolated by laser cell microdissection (LCM), and RNA was extracted with the RNeasy FFPE Kit. RNA concentration was measured using a NanoDrop 2000, a Qubit 3.0 fluorimeter with the Qubit RNA HS Assay, and a Bioanalyzer 2100 with the Agilent RNA 6000 Pico Kit. RNA quality was assessed by RNA integrity number (RIN) and DV200 value (percentage of fragments > 200 nt) using a Bioanalyzer 2100; the authors also calculated the quantity of RNA > 200 nt per unit area by multiplying the DV200 value by the total RNA amount determined with a bioanalyzer. For NGS, library preparation and sequencing were performed using the Oncomine Dx Target Test Multi-CDx System with the RNA panel; the total number of mapped reads and read counts of internal control genes (HMBS, ITGB7, LRP1, MYC, TBP) were used to assess differences between tissue blocks fixed for different durations. Library concentration was measured using the Ion Library Quantitation Kit with real-time PCR prior to equalization with the Ion PGM Dx Library Equalizer Kit.
Summary of Findings:
In the one HCC specimen analyzed, total RNA yield declined with fixation duration (1-240 h) for all three quantification methods (Qubit, NanoDrop, Bioanalyzer). The yield of RNA per unit of LCM tissue surface area was greatest when the HCC specimen was fixed in formalin for 2 h at room temperature compared to fixation for longer durations for all quantification methods, but RNA yield was the greatest in the 2 h specimen when RNA was quantified from the specimen by bioanalyzer (89.2 ng/mm2), followed by Nanodrop (56.1 ng/mm) and Qubit (53.7 ng/mm2). Comparatively, the HCC tissue block fixed for 48 h at room temperature displayed a reduction in total RNA to just 43, 38, and 24% of the yield observed in the tissue block fixed for 2 h when quantified by NanoDrop, Qubit, and bioanalyzer, respectively. While DV200 values displayed variability during the time course, DV200 values tended to decrease and a clear progressive decline in the total amount of RNA > 200 nt was observed with longer fixation in comparison to the control specimen fixed for 1 h; RIN was not affected by the duration of fixation. Library concentration (prior to equalization) was negatively correlated with the duration of fixation (r= - 0.9181), and tissue blocks that were fixed longer than 72 h yielded libraries below the recommended concentration prior to equalizing (100 pmol/L). Although the duration of formalin fixation was not correlated to the total number of mapped reads (r = - 0.12), it was negatively correlated to the number of mapped reads for each of the following internal control genes: HMBS (r = -0.79), ITGB7 (r= -0.79), and MYC (r= -0.89). When FFPE blocks fixed for different durations were stored for 500 d at room temperature, the total amount of RNA per tissue area (determined by bioanalyzer) was reduced for 8 of the 11 fixation duration timepoints (1, 2, 4, 6, 48, 72, 120, 168 h) compared to values when the blocks were analyzed immediately. Storage of FFPE blocks containing tissue that was fixed for 1-12 h resulted in lower DV200 values and amounts of total RNA and RNA fragments >200 nt compared to values when the same tissue was analyzed immediately.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Fluorometry RNA Spectrophotometry RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 1 h
2 h
4 h
6 h
12 h
24 h
48 h
72 h
120 h
168 h
240 h
Automated electrophoresis/Bioanalyzer Specific Quality metrics RIN
DV200
Storage Storage duration Analyzed immediately
500 days