The Paraffin-Embedded RNA Metric (PERM) for RNA Isolated from Formalin-Fixed, Paraffin-Embedded Tissue

Chung, Joon‐Yong; Cho, Hanbyoul; Hewitt, Stephen M.

doi:10.2144/000114415

Cited by 19 publications

(19 citation statements)

References 16 publications

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“…Recently, paraffin-embedded RNA metrics (PERM) is also proposed as a novel indicator that is based on the intensity of fluorescence at specific time points using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) [10]. Although we attempted to perform a PERM analysis, unfortunately, the TapeStation used in this study did not support the PERM analysis.…”

Section: Discussionmentioning

confidence: 95%

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

Matsubara

Soh

Morita

et al. 2020

BioMed Research International

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Poor quality of biological samples will result in an inaccurate analysis of next-generation sequencing (NGS). Therefore, methods to accurately evaluate sample integrity are needed. Among methods for evaluating RNA quality, the RNA integrity number equivalent (RINe) is widely used, whereas the DV200, which evaluates the percentage of fragments of >200 nucleotides, is also used as a quality assessment standard. In this study, we compared the RINe and DV200 RNA quality indexes to determine the most suitable RNA index for the NGS analysis. Seventy-one RNA samples were extracted from formalin-fixed paraffin-embedded tissue samples (n = 30), fresh-frozen samples (n = 25), or cell lines (n = 16). After assessing RNA quality using the RINe and DV200, we prepared two kinds of stranded mRNA sequencing libraries. Finally, we calculated the correlation between each RNA quality index and the amount of library product (1 st PCR product per input RNA). The DV200 measure showed stronger correlation with the amount of library product than the RINe (R 2 = 0:8208 for the DV200 versus 0.6927 for the RINe). Receiver operating characteristic curve analyses revealed that the DV200 was the better marker for predicting efficient library production than the RINe using a threshold of >10 ng/ng for the amount of the 1 st PCR product per input RNA (cutoff value for the RINe and DV200, 2.3 and 66.1%; area under the curve, 0.99 and 0.91; sensitivity, 82% and 92%; and specificity, 93% and 100%, respectively). Our results indicate that NGS libraries prepared using RNA samples with the DV200 value > 66:1% exhibit greater sensitivity and specificity than those prepared with the RINe values > 2:3. These findings suggest that the DV200 is superior to the RINe, especially for low-quality RNA, because it is a more consistent assessment of the amount of the 1 st NGS library product per input.

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Section: Discussionmentioning

confidence: 95%

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

Matsubara

Soh

Morita

et al. 2020

BioMed Research International

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“…Fixation and paraffin-embedding of tissue allows for long term preservation and storage of samples while retaining useful morphological information. The process of fixation, embedding, and extraction can damage RNA, and long-term storage of such samples can make the damage progressively worse, making gene expression analysis difficult (4,13). However, due to the nature of DO hybridization and ligation chemistry and the short length of RNA sequence that is targeted by each DO set, TempO-Seq is highly resistant to this type of fragmentation, as well as to the presence of crosslinking.…”

Section: Archival Tissuementioning

confidence: 99%

Extraction-Free Whole Transcriptome Gene Expression Analysis of FFPE Sections and Histology-Directed Subareas of Tissue

Trejo

Babic

Imler

et al. 2018

Preprint

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We describe the use of a ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq™, to profile formalin-fixed paraffin-embedded (FFPE) tissue, including H&E stained FFPE tissue, by directly lysing tissue scraped from slides without extracting RNA or converting the RNA to cDNA. The correlation of measured gene expression changes in unfixed and fixed samples using blocks prepared from a pellet of a single cell type was R 2 = 0.97, demonstrating that no significant artifacts were introduced by fixation. Fixed and fresh samples prepared in an equivalent manner produced comparable sequencing depth results (+/-20%), with similar %CV (11.5 and 12.7%, respectively), indicating no significant loss of measurable RNA due to fixation. The sensitivity of the TempO-Seq assay was the same whether the tissue section was fixed or not. The assay performance was equivalent for human, mouse, or rat whole transcriptome. The results from 10 mm 2 and 2 mm 2 areas of tissue obtained from 5 μm thick sections were equivalent, thus demonstrating high sensitivity and ability to profile focal areas of histology within a section. Replicate reproducibility of separate areas of tissue ranged from R 2 = 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R 2 = 0.90 across nine tissue types. The average %CVs were 16.8% for genes expressed at greater than 200 counts, and 20.3% for genes greater than 50 counts. Tissue specific differences in gene expression were identified and agreed with the literature. There was negligible impact on assay performance using FFPE tissues that had been archived for up to 30 years. Similarly, there was negligible impact of H&E staining, facilitating accurate visualization for scraping and assay of small focal areas of specific histology within a section.

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“…In addition, we also assessed RNA integrity by the paraffin-embedded RNA metric (PERM) number which is a novel metric for FFPE RNA. 21 The extracted RNA was treated with 2 μl TURBO DNase buffer, 4 units TURBO DNase (Invitrogen), and 40 units of RNase inhibitor (Promega, Madison, WI, USA) in a 100 μl reaction volume to remove possible contaminating genomic DNA. Reverse transcription was performed with 5 μg total RNA using Superscript II RNA H-Reverse Transcriptase (Invitrogen).…”

Section: Protein Extraction and Western Blottingmentioning

confidence: 99%

“…21 The mean PERM number of 0.5% H 2 O 2 with Tris-HCl (36.5 ± 0.4, Po0.001) and 0.5% H 2 O 2 with PBS (31. 7 ± 1.4, P = 0.021) was significantly higher than that of the non-bleached condition (28.2 ± 1.3).…”

Section: Rna Integrity Was Retained In the Bleached Ffpe Tissuementioning

confidence: 99%

A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue

Chung

Choi

Sears

et al. 2016

Laboratory Investigation

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Removal of excessive melanin from heavily pigmented formalin-fixed paraffin-embedded (FFPE) melanoma tissues is essential for histomorphological and molecular diagnostic assessments. Although there have been efforts to address this issue, current methodologies remain complex and time-consuming, and are not suitable for multiple molecular applications. Herein, we have developed a robust and rapid melanin-bleaching methodology for FFPE tissue specimens. Our approach is based on quick bleaching (15 min) at high temperature (80°C) with 0.5% diluted hydrogen peroxide (H 2 O 2 ) in Tris-HCl, PBS, or Tris/Tricine/SDS buffer. Immunostaining for Ki-67 and HMB45 was enhanced by bleaching with 0.5% H 2 O 2 in Tris/Tricine/SDS and Tris-HCl, respectively. In addition to histopathological applications, our approach also facilitates recovery of protein and nucleic acid from archival melanin-rich FFPE tissue sections. Protein extracted from bleached FFPE tissues was compatible with western blotting using anti-human GAPDH and AKT antibodies. Our bleaching condition significantly improved RNA quality compared with unbleached tissues without compromising the yield. Notably, the RNA/DNA obtained from bleached tissues was suitable for end point PCR and real-time quantitative RT-PCR. In conclusion, this improved melanin-bleaching method enhances and simplifies immunostaining procedures, and facilitates the use of melanin-rich FFPE tissues for histomorphological and PCR amplification-based molecular assays.

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The Paraffin-Embedded RNA Metric (PERM) for RNA Isolated from Formalin-Fixed, Paraffin-Embedded Tissue

Cited by 19 publications

References 16 publications

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

Extraction-Free Whole Transcriptome Gene Expression Analysis of FFPE Sections and Histology-Directed Subareas of Tissue

A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue

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