Cargando…
Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens
BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separa...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326040/ https://www.ncbi.nlm.nih.gov/pubmed/22514653 http://dx.doi.org/10.1371/journal.pone.0034683 |
_version_ | 1782229496482496512 |
---|---|
author | Kotorashvili, Adam Ramnauth, Andrew Liu, Christina Lin, Juan Ye, Kenny Kim, Ryung Hazan, Rachel Rohan, Thomas Fineberg, Susan Loudig, Olivier |
author_facet | Kotorashvili, Adam Ramnauth, Andrew Liu, Christina Lin, Juan Ye, Kenny Kim, Ryung Hazan, Rachel Rohan, Thomas Fineberg, Susan Loudig, Olivier |
author_sort | Kotorashvili, Adam |
collection | PubMed |
description | BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. PRINCIPAL FINDINGS: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SIGNIFICANCE: We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies. |
format | Online Article Text |
id | pubmed-3326040 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-33260402012-04-18 Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens Kotorashvili, Adam Ramnauth, Andrew Liu, Christina Lin, Juan Ye, Kenny Kim, Ryung Hazan, Rachel Rohan, Thomas Fineberg, Susan Loudig, Olivier PLoS One Research Article BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. PRINCIPAL FINDINGS: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SIGNIFICANCE: We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies. Public Library of Science 2012-04-13 /pmc/articles/PMC3326040/ /pubmed/22514653 http://dx.doi.org/10.1371/journal.pone.0034683 Text en Kotorashvili 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 Kotorashvili, Adam Ramnauth, Andrew Liu, Christina Lin, Juan Ye, Kenny Kim, Ryung Hazan, Rachel Rohan, Thomas Fineberg, Susan Loudig, Olivier Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens |
title | Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens |
title_full | Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens |
title_fullStr | Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens |
title_full_unstemmed | Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens |
title_short | Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens |
title_sort | effective dna/rna co-extraction for analysis of micrornas, mrnas, and genomic dna from formalin-fixed paraffin-embedded specimens |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326040/ https://www.ncbi.nlm.nih.gov/pubmed/22514653 http://dx.doi.org/10.1371/journal.pone.0034683 |
work_keys_str_mv | AT kotorashviliadam effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens AT ramnauthandrew effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens AT liuchristina effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens AT linjuan effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens AT yekenny effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens AT kimryung effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens AT hazanrachel effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens AT rohanthomas effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens AT finebergsusan effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens AT loudigolivier effectivednarnacoextractionforanalysisofmicrornasmrnasandgenomicdnafromformalinfixedparaffinembeddedspecimens |