Transparent DNA/RNA co-extraction workflow protocol suitable for inhibitor-rich environmental samples that focuses on complete DNA removal for transcriptomic analyses
Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultan...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2016-10-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.01588/full |
| _version_ | 1819031815410679808 |
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| author | Natalie Yen Nee Lim Constance Armanda Roco Constance Armanda Roco Asa Frostegard |
| author_facet | Natalie Yen Nee Lim Constance Armanda Roco Constance Armanda Roco Asa Frostegard |
| author_sort | Natalie Yen Nee Lim |
| collection | DOAJ |
| description | Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer’s recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of representative samples is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis. |
| first_indexed | 2024-12-21T06:52:03Z |
| format | Article |
| id | doaj.art-b4b1486551fa426fa69c8bcac5e1dd15 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-21T06:52:03Z |
| publishDate | 2016-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-b4b1486551fa426fa69c8bcac5e1dd152022-12-21T19:12:27ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2016-10-01710.3389/fmicb.2016.01588213819Transparent DNA/RNA co-extraction workflow protocol suitable for inhibitor-rich environmental samples that focuses on complete DNA removal for transcriptomic analysesNatalie Yen Nee Lim0Constance Armanda Roco1Constance Armanda Roco2Asa Frostegard3Norwegian University of Life SciencesCornell UniversityNorwegian University of Life SciencesNorwegian University of Life SciencesAdequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer’s recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of representative samples is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis.http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.01588/fullpurificationenzyme inhibitionRNA ExtractionEnvironmental sampleGenomic DNA removal |
| spellingShingle | Natalie Yen Nee Lim Constance Armanda Roco Constance Armanda Roco Asa Frostegard Transparent DNA/RNA co-extraction workflow protocol suitable for inhibitor-rich environmental samples that focuses on complete DNA removal for transcriptomic analyses Frontiers in Microbiology purification enzyme inhibition RNA Extraction Environmental sample Genomic DNA removal |
| title | Transparent DNA/RNA co-extraction workflow protocol suitable for inhibitor-rich environmental samples that focuses on complete DNA removal for transcriptomic analyses |
| title_full | Transparent DNA/RNA co-extraction workflow protocol suitable for inhibitor-rich environmental samples that focuses on complete DNA removal for transcriptomic analyses |
| title_fullStr | Transparent DNA/RNA co-extraction workflow protocol suitable for inhibitor-rich environmental samples that focuses on complete DNA removal for transcriptomic analyses |
| title_full_unstemmed | Transparent DNA/RNA co-extraction workflow protocol suitable for inhibitor-rich environmental samples that focuses on complete DNA removal for transcriptomic analyses |
| title_short | Transparent DNA/RNA co-extraction workflow protocol suitable for inhibitor-rich environmental samples that focuses on complete DNA removal for transcriptomic analyses |
| title_sort | transparent dna rna co extraction workflow protocol suitable for inhibitor rich environmental samples that focuses on complete dna removal for transcriptomic analyses |
| topic | purification enzyme inhibition RNA Extraction Environmental sample Genomic DNA removal |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.01588/full |
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