Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue.
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...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2019-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0212031 |
| _version_ | 1818989420722782208 |
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| author | Christy L Trejo Miloš Babić Elliot Imler Migdalia Gonzalez Sergei I Bibikov Peter J Shepard Harper C VanSteenhouse Joanne M Yeakley Bruce E Seligmann |
| author_facet | Christy L Trejo Miloš Babić Elliot Imler Migdalia Gonzalez Sergei I Bibikov Peter J Shepard Harper C VanSteenhouse Joanne M Yeakley Bruce E Seligmann |
| author_sort | Christy L Trejo |
| collection | DOAJ |
| description | 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 R2 = 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 mm2 and 2 mm2 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 R2 = 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R2 = 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. |
| first_indexed | 2024-12-20T19:38:12Z |
| format | Article |
| id | doaj.art-24977ed9ec084aca885a049108f285f0 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-20T19:38:12Z |
| publishDate | 2019-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-24977ed9ec084aca885a049108f285f02022-12-21T19:28:35ZengPublic Library of Science (PLoS)PLoS ONE1932-62032019-01-01142e021203110.1371/journal.pone.0212031Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue.Christy L TrejoMiloš BabićElliot ImlerMigdalia GonzalezSergei I BibikovPeter J ShepardHarper C VanSteenhouseJoanne M YeakleyBruce E SeligmannWe 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 R2 = 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 mm2 and 2 mm2 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 R2 = 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R2 = 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.https://doi.org/10.1371/journal.pone.0212031 |
| spellingShingle | Christy L Trejo Miloš Babić Elliot Imler Migdalia Gonzalez Sergei I Bibikov Peter J Shepard Harper C VanSteenhouse Joanne M Yeakley Bruce E Seligmann Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue. PLoS ONE |
| title | Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue. |
| title_full | Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue. |
| title_fullStr | Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue. |
| title_full_unstemmed | Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue. |
| title_short | Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue. |
| title_sort | extraction free whole transcriptome gene expression analysis of ffpe sections and histology directed subareas of tissue |
| url | https://doi.org/10.1371/journal.pone.0212031 |
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