Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples
BACKGROUND: We have developed a gene expression assay (Whole-Genome DASL®), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated a similar level of sensitivity in...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2009
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2780295/ https://www.ncbi.nlm.nih.gov/pubmed/19997620 http://dx.doi.org/10.1371/journal.pone.0008162 |
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author | April, Craig Klotzle, Brandy Royce, Thomas Wickham-Garcia, Eliza Boyaniwsky, Tanya Izzo, John Cox, Donald Jones, Wendell Rubio, Renee Holton, Kristina Matulonis, Ursula Quackenbush, John Fan, Jian-Bing |
author_facet | April, Craig Klotzle, Brandy Royce, Thomas Wickham-Garcia, Eliza Boyaniwsky, Tanya Izzo, John Cox, Donald Jones, Wendell Rubio, Renee Holton, Kristina Matulonis, Ursula Quackenbush, John Fan, Jian-Bing |
author_sort | April, Craig |
collection | PubMed |
description | BACKGROUND: We have developed a gene expression assay (Whole-Genome DASL®), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3–1.5 and 1.5–2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is ∼3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) ∼0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2)∼0.86 and R(2)∼0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2)∼0.92), with ∼71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2)∼0.80, while still maintaining a correlation of R(2)∼0.75 with standard FFPE inputs (200 ng). CONCLUSIONS/SIGNIFICANCE: Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens). |
format | Text |
id | pubmed-2780295 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-27802952009-12-08 Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples April, Craig Klotzle, Brandy Royce, Thomas Wickham-Garcia, Eliza Boyaniwsky, Tanya Izzo, John Cox, Donald Jones, Wendell Rubio, Renee Holton, Kristina Matulonis, Ursula Quackenbush, John Fan, Jian-Bing PLoS One Research Article BACKGROUND: We have developed a gene expression assay (Whole-Genome DASL®), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3–1.5 and 1.5–2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is ∼3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) ∼0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2)∼0.86 and R(2)∼0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2)∼0.92), with ∼71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2)∼0.80, while still maintaining a correlation of R(2)∼0.75 with standard FFPE inputs (200 ng). CONCLUSIONS/SIGNIFICANCE: Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens). Public Library of Science 2009-12-03 /pmc/articles/PMC2780295/ /pubmed/19997620 http://dx.doi.org/10.1371/journal.pone.0008162 Text en April et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article April, Craig Klotzle, Brandy Royce, Thomas Wickham-Garcia, Eliza Boyaniwsky, Tanya Izzo, John Cox, Donald Jones, Wendell Rubio, Renee Holton, Kristina Matulonis, Ursula Quackenbush, John Fan, Jian-Bing Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples |
title | Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples |
title_full | Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples |
title_fullStr | Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples |
title_full_unstemmed | Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples |
title_short | Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples |
title_sort | whole-genome gene expression profiling of formalin-fixed, paraffin-embedded tissue samples |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2780295/ https://www.ncbi.nlm.nih.gov/pubmed/19997620 http://dx.doi.org/10.1371/journal.pone.0008162 |
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