Method Based on Electrophoresis and Gel Extraction for Obtaining Genomic DNA-free cDNA Without DNase Treatment
Year: 2004
Type: article
Abstract: BioTechniquesVol. 37, No. 5 BenchmarksOpen AccessMethod based on electrophoresis and gel extraction for obtaining genomic DNA-free cDNA without DNase treatmentLaura Jaakola, Anna Maria Pirttilä, Jaana Vuosku & Anja HohtolaLaura Jaakola*Address correspondence to: Laura Jaakola, Department of Biology/Botany, University of Oulu, P.O. Box 3000, FIN-90014 Oulu, Finland. e-mail: E-mail Address: [email protected] of Oulu, Oulu, Finland, Anna Maria PirttiläUniversity of Oulu, Oulu, Finland, Jaana VuoskuUniversity of Oulu, Oulu, Finland & Anja HohtolaUniversity of Oulu, Oulu, FinlandPublished Online:6 Jun 2018https://doi.org/10.2144/04375BM06AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail For studying differential gene expression by reverse-transcriptase PCR (RT-PCR) applications, eliminating genomic DNA contamination from the cDNA is essential prior to amplification of the specific gene sequence. Residual genomic DNA can disturb the amplification of the target gene from cDNA and lead to false-positive results. DNase I treatment is generally used to remove genomic DNA from RNA samples. However, RNA can degrade totally or partially during the DNase treatment. It has also been shown that some contamination of genomic DNA can remain after the DNase treatment, even after an overnight incubation (1). Certain tissue types can be a problem because some contain elevated levels of RNases (2,3) or a limited amount of RNA (4). Fruit tissues are regarded as particularly problematic because of high amounts of secondary metabolites, polysaccharides, and elevated levels of RNases (5). In our experiments with RNA from different plant tissues, especially bilberry (Vaccinium myrtillus) fruit and leaf, we found that RNA degraded during the DNase treatment. We also noticed that despite the DNase treatment, some genomic DNA was always left in the samples when RT-PCR gene expression analysis was performed on Scots pine (Pinus sylvestris) needles. To overcome these problems, we developed a method based on electrophoresis and gel-extraction filter tubes.Bilberry fruits and leaves and Scots pine needles growing in a forest in Oulu, Finland, were snap frozen in liquid nitrogen and stored at °80°C until use. Total RNA was isolated from the samples with the method described for pine trees (6) that had been modified by Jaakola et al. (7). The quality of the isolated RNA was verified on a 1% (w/v) ethidium bromide-stained agarose gel and from the absorbance measurements at wavelengths 230, 260, and 280 nm.From each sample, 5 µg were taken for DNase treatment with TURBO™ DNase (Ambion, Austin, TX, USA), according to the manufacturer's instructions (1 U/1 µg DNA) using SUPER-ase-In™ RNase inhibitor (Ambion) in a reaction volume of 50 µL at 37°C for 30 min. Figure 1A shows the isolated RNA before and after the DNase treatment. As seen in the figure, RNA from bilberry fruits (lane 6) is totally degraded, and RNA from leaf sample (lane 5) is mostly degraded after the DNase treatment. The RNA from pine needles is still intact, but the amount has been reduced to 1/5 of the original.Figure 1. Preparation of genomic DNA-free cDNA.(A) Separation of isolated total RNA (0.5 µg) by electrophoresis before (lanes 1–3) and after (lanes 4–6) DNase treatment. Lanes 1 and 4: pine needle RNA; lanes 2 and 5: bilberry leaf RNA; lanes 3 and 6: bilberry fruit RNA. (B) Separation of cDNA from genomic DNA prior to centrifugation at 5000× g with gel-extraction tubes. Lane 1: pine needle cDNA; lane 2: bilberry leaf cDNA; lane 3: bilberry fruit cDNA. (C) Amplification using glyceraldehyde-3-phosphate dehydrogenase (GAPD)/pine primers of (lane 1) pine cDNA and (lane 2) pine genomic DNA; amplification using GAPD/bilberry primers of (lane 3) bilberry leaf cDNA, (lane 4) bilberry fruit cDNA, and (lane 5) bilberry genomic DNA. (D) Lanes 1, 4, and 7: gel-separated pine cDNA; lanes 2, 5, and 8: pine cDNA without separation; lanes 3, 6, and 9: pine genomic DNA. Lanes 1–3: PCR with actin (ACT) primers; lanes 4–6: PCR with chalcone synthase (CHS) primers; lanes 7–9: PCR with pinosylvin synthase (BBS) primers. In all cases, lane M corresponds to GeneRuler™ DNA Ladder Mix (Fermentas, Vilnius, Lithuania).In addition, 5 µg of each sample of non-DNase-treated RNA were reverse transcribed using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with anchored oligo(dT) primers using standard methods in a reaction volume of 20 µL. The samples were separated for 15 min at 40 volts (V) on a 1% ethidium bromide-stained agarose gel prepared in Tris-acetate-EDTA (TAE) buffer. The cDNA fragments were trimmed out of the gel according to the size information (<10 kb), leaving the genomic DNA on the gel (Figure 1B). The fragments were extracted from agarose gel slices with the Montage® DNA Gel Extraction Kit (Millipore, Bedford, MA, USA), which is designed to extract DNA fragments that are 100 to 10,000 bp in size in one 10-min centrifugation at 5000× g. After the centrifugation, the solution containing the cDNA was precipitated by adding two volumes of ice-cold absolute ethanol, incubating at -70°C for 30 min, and centrifuging at 18,000× g for 15 min at 4°C. The pellet was suspended in 10 µL sterilized water.For verifying the purity of the cDNA from genomic DNA, PCR was performed using the gel-purified cDNA or genomic DNA as template. For the latter, DNA was isolated from pine needles and bilberry leaves with the method described by Pirttilä et al. (8). DyNA-zyme™ DNA polymerase (Finnzymes, Espoo, Finland) was used for the PCR amplification in a final volume of 25 µL. Conditions for PCR for pine and bilberry cDNA and DNA were 30 cycles of 94°C for 60 s, 54°–63°C for 2 min, and 72°C for 2 min. Table 1 presents the annealing temperature for each primer. The PCR products were separated on a 1% (w/v) agarose gel stained with ethidium bromide. Precise sizes for expected amplification products are given in Table 1; in all cases, the primer pairs encompass a region containing at least one intron, thus allowing cDNA and genomic DNA amplification products to be distinguished. As a first analysis, primer pairs against glyceraldehyde-3-phosphate dehydrogenase (GAPD) were used to amplify products from pine and bilberry cDNA and DNA samples (Figure 1C). No bands indicative of genomic DNA contamination were seen in the gel-purified cDNA samples. To confirm the importance of the purification step, additional primer pairs against actin (ACT), chalcone synthase (CHS), and pinosylvin synthase (BBS) were used for amplifying fragments from pine cDNA samples with and without gel separation and from the genomic DNA as a control (Figure 1D). In samples without gel separation, using CHS and BBS primers, the amplification occurred primarily from genomic DNA instead of cDNA. With ACT primers, both cDNA and genomic DNA were equally amplified from the untreated samples (Figure 1D). These results clearly show how the presence of genomic DNA disturbs the amplification of a target gene from cDNA. In quantitative RT-PCR, the results would thus be greatly distorted.Table 1. Sequences of the Primers and Information of the Reaction Conditions and Amplification ProductsThe elimination of genomic DNA from RNA or cDNA is required to study gene expression with RT-PCR or micro-arrays. A common strategy for removing genomic DNA is DNase treatment; however, typical problems include the degradation of the RNA and the presence of residual contaminating genomic DNA. Our results show that the method described here is suitable for obtaining intact and functional cDNA that is free of genomic DNA. We have found this method useful with all RNA samples, but especially with samples that degrade easily during the DNase treatment step. The method is efficient because no time-consuming extraction steps with hazardous chemicals are needed. Flohr et al. (9) reported the possibility of performing the DNase treatment on cDNA instead of RNA, which is also an alternative for the RNA samples that degrade easily during the DNase treatment. However, because of the extraction steps, some loss of the sample always occurs during the DNase treatment. Although some reduction in sample amount is also encountered during the gel-extraction step, we have found this method more reliable because residual genomic DNA was frequently detected when DNase treatment was used.AcknowledgmentsThis work was supported by Academy of Finland, SUNARE project (Sustainable Use of Natural Resources) grant no. 52741, and by the Emil Aaltonen Foundation.Competing Interests StatementThe authors declare no competing interests.References1. Ivarsson, K. and B. Weijdegard. 1998. Evaluation of the effects of DNase treatment on signal specifity in RT-PCR and in situ RTPCR. BioTechniques 25:630–638.Link, CAS, Google Scholar2. Groppe, J.C. and D.E. Morse. 1993. Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans. Anal. Biochem. 210:337–343.Crossref, Medline, CAS, Google Scholar3. Pillon, D. and G. Bruneau. 2003. Resistant ribonuclease activity in preparations of total RNA extracted from artiodactylbrain with GITC. BioTechniques 34:920–924.Link, CAS, Google Scholar4. Miller, C.L. and R.H. Yolken. 2003. Methods to optimize the generation of cDNA from postmortem human brain tissue. Brain Res. Prot. 10:156–167.Crossref, Medline, CAS, Google Scholar5. Rodrigues-Pousada, R., M. Van Montagu, and D. Van der Straeten. 1990. A protocol for preparation of total RNA from fruit. Technique 2:292–294.CAS, Google Scholar6. Chang, S., J. Puryear, and J. Cairney. 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Reptr. 11:113–116.Crossref, CAS, Google Scholar7. Jaakola, L., A.M. Pirttilä, M. Halonen, and A. Hohtola. 2001. Isolation of high quality RNA from the bilberry (Vaccinium myrtillus L.) fruit. Mol. Biotechnol. 19:201–203.Crossref, Medline, CAS, Google Scholar8. Pirttilä, A.M., T. Kämäräinen, M. Hirsikorpi, L. Jaakola, and A. Hohtola. 2001. A DNA isolation method for medicinal and aromatic plants. Plant Mol. Biol. Reptr. 19:273a–f.Crossref, Google Scholar9. Flohr, A.M., T. Hackenbeck, C. Schlueter, P. Rogalla, and J. Bullerdiek. 2003. Dnase I treatment of cDNA first strands prevents RTPCR amplification of contaminating DNA sequences. BioTechniques 35:920–926.Link, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByHost species shape the community structure of culturable endophytes in fruits of wild berry species ( Vaccinium myrtillus L., Empetrum nigrum L. and Vaccinium vitis-idaea L.)12 July 2021 | FEMS Microbiology Ecology, Vol. 97, No. 8Changes in the Proanthocyanidin Composition and Related Gene Expression in Bilberry ( Vaccinium myrtillus L.) Tissues16 June 2020 | Journal of Agricultural and Food Chemistry, Vol. 68, No. 28Thermospermine Synthase (ACL5) and Diamine Oxidase (DAO) Expression Is Needed for Zygotic Embryogenesis and Vascular Development in Scots Pine20 December 2019 | Frontiers in Plant Science, Vol. 10Abscisic acid is involved in phenolic compounds biosynthesis, mainly anthocyanins, in leaves of Aristotelia chilensis plants (Mol.) subjected to drought stress10 September 2018 | Physiologia Plantarum, Vol. 165, No. 4Recognition of candidate transcription factors related to bilberry fruit ripening by de novo transcriptome and qRT-PCR analyses2 July 2018 | Scientific Reports, Vol. 8, No. 1Natural variation of DNA methylation and gene expression may determine local adaptations of Scots pine populations3 August 2018 | Journal of Experimental Botany, Vol. 69, No. 21Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits29 August 2018 | Frontiers in Plant Science, Vol. 9Age-related mechanism and its relationship with secondary metabolism and abscisic acid in Aristotelia chilensis plants subjected to drought stressPlant Physiology and Biochemistry, Vol. 124Different levels of UV-B resistance in Vaccinium corymbosum cultivars reveal distinct backgrounds of phenylpropanoid metabolitesPlant Physiology and Biochemistry, Vol. 118Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation21 April 2016 | BMC Plant Biology, Vol. 16, No. 1Molecular Cloning and Expression Analysis of hyp-1 Type PR-10 Family Genes in Hypericum perforatum21 April 2016 | Frontiers in Plant Science, Vol. 7Moderate stress responses and specific changes in polyamine metabolism characterize Scots pine somatic embryogenesis19 January 2016 | Tree Physiology, Vol. 36, No. 3Metabolic and molecular analyses of white mutant Vaccinium berries show down-regulation of MYBPA1-type R2R3 MYB regulatory factor14 July 2015 | Planta, Vol. 242, No. 3Hypericum perforatum hydroxyalkylpyrone synthase involved in sporopollenin biosynthesis - phylogeny, site-directed mutagenesis, and expression in nonanther tissues28 July 2014 | FEBS Journal, Vol. 281, No. 17Changes in the abscisic acid levels and related gene expression during fruit development and ripening in bilberry (Vaccinium myrtillus L.)Phytochemistry, Vol. 95Ascorbic acid metabolism during bilberry (Vaccinium myrtillus L.) fruit developmentJournal of Plant Physiology, Vol. 169, No. 11Biofilm formation and virulence of uropathogenic Escherichia coli in urine after consumption of cranberry-lingonberry juice7 August 2011 | European Journal of Clinical Microbiology & Infectious Diseases, Vol. 31, No. 5Endophytic bacteria enhancing growth and disease resistance of potato (Solanum tuberosum L.)Biological Control, Vol. 56, No. 1Isolation and genotype-dependent, organ-specific expression analysis of a Rhodiola rosea cDNA encoding tyrosine decarboxylaseJournal of Plant Physiology, Vol. 166, No. 14Molecular cloning and tissue-specific expression of two cDNAs encoding polyketide synthases from Hypericum perforatumJournal of Plant Physiology, Vol. 165, No. 10Consistency of Polyamine Profiles and Expression of Arginine Decarboxylase in Mitosis during Zygotic Embryogenesis of Scots Pine8 September 2006 | Plant Physiology, Vol. 142, No. 3 Vol. 37, No. 5 Follow us on social media for the latest updates Metrics Downloaded 1,967 times History Received 29 April 2004 Accepted 13 July 2004 Published online 6 June 2018 Published in print November 2004 Information© 2004 Author(s)AcknowledgmentsThis work was supported by Academy of Finland, SUNARE project (Sustainable Use of Natural Resources) grant no. 52741, and by the Emil Aaltonen Foundation.Competing Interests StatementThe authors declare no competing interests.PDF download
Source: BioTechniques
DOI: 10.2144/04375bm06
Institution: University of Oulu
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,415
Funder: Academy of Finland
Award: G8148345625
OpenAlex ID: W39299196