Simultaneous DNA and RNA isolation from brain punches for epigenetics
© Murgatroyd et al; licensee BioMed Central Ltd. 2011
Received: 25 March 2011
Accepted: 30 August 2011
Published: 30 August 2011
Epigenetic modifications such as DNA methylation play an important role for gene expression and are regulated by developmental and environmental signals. DNA methylation typically occurs in a highly tissue- and cell-specific manner. This raises a severe challenge when studying discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Because gene expression and methylation are often tightly linked it appears of interest to compare both parameters in the same sample.
We present a refined method for the simultaneous extraction of DNA for bisulfite sequencing and RNA for expression analysis from small mouse brain tissue punches. This method can also be easily adapted for other small tissues or cell populations.
The method described herein results in DNA and RNA of a quantity and quality permitting highly reliable bisulfite analysis and quantitative RT-PCR measurements, respectively.
The spatio-temporal expression of a gene is defined by DNA sequence (per se) and the manner by which it is marked through epigenetic mechanisms including DNA methylation and chromatin modification. In eukaryotes DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosines that are followed by guanines, i.e. CpG dinucleotides. Functionally, DNA methylation frequently confers gene silencing.
CpG methylation of genomic DNA is routinely analyzed by the treatment of DNA with sodium bisulfite, followed by PCR amplification and sequencing . While bisulfite readily deaminates cytosine residues to uracils, which are then converted to thymines during DNA amplification by PCR, 5-methylcytosine resists this modification. Many methods based on this principle have been developed including direct sequencing, pyrosequencing, methylation-specific PCR (MSP), combined bisulfite restriction analysis (COBRA), methylation-sensitive single nucleotide primer extension (MS-SNuPE) and microarray-based methods (for review see ).
Bisulfite analysis depends on high quantity and quality of DNA as the bisulfite conversion procedure itself requires long incubation times, elevated temperature, and high bisulfite concentration; all of which are highly detrimental to DNA . Furthermore, to investigate the functional interrelationship between DNA methylation and RNA expression both should be determined within the same sample. In this respect, the analysis of expression data and DNA methylation from two separate cohorts of animals may introduce a bias, unless at least double the numbers of animals are included in each cohort. Similarly, the surgical splitting of tissues containing different cell types can confound the analysis as DNA methylation is highly tissue- and even cell-type specific. Finally, tissue punches of usually around 0.8 mm from distinct areas of the brain, are generally rather limiting.
Though a number of different methods have been developed for simultaneous extraction of DNA and RNA, a technique addressing efficient isolation from small tissue samples has not been reported so far. While TRIzol can be used for the simultaneous extraction of DNA and RNA, in addition to proteins , we note that that the quality of DNA produced from small tissues was not high enough for bisulfite analysis. Furthermore, we find that commercially available kits for RNA/DNA isolation, relying on spin-column purification , do not yield a high enough DNA quantity from small tissues to permit reliable bisulfite analysis (data not shown).
We have therefore adapted a derivative of the guanidinium thiocyanate-phenol-chloroform extraction method, originally devised by Piotr Chomczynski and Nicoletta Sacchi for the extraction of RNA . While variants of a guanidinium thiocyanate-based (GTC) buffer have been used for RNA (for review see ), various forms of a guanidinium thiocyanate-based buffer have also proved efficient for the purification of DNA [8–12] and can be further modified for the simultaneous extraction of RNA and DNA in cancer tissues  and whole fish embryos . Here we describe our experience in extracting both DNA and RNA from punched brain tissue and present an alternative for obtaining both DNA and RNA from the same cells for genome and transcriptome profiling. In addition, we characterized tissue specimens and cell quantities needed for this method.
Materials and Methods
Yield and purity of DNA and RNA preparations from 0,8 mm cortex punches using commonly employed protocols, commercially available kits and the presented simultaneous DNA/RNA extraction method
Total yield [ng]
(n = 6)
360 ± 96
520 ± 87
850 ± 195
360 ± 96
(n = 6)
MN Nucleospin II
366 ± 75
690 ± 262
838 ± 321
(n = 9)
765 ± 163
330 ± 54
RNA/DNA yield, A260/280 and 260/230 ratios for the extracted samples were analyzed with an Implen Nanophotometer.
Yields of simultaneously isolated RNA and DNA from brain tissue punches (0,8 mm) of different brain regions and whole pituitary
Total DNA yield [ng]
Total RNA yield [ng]
PVN (n = 10)
DG (n = 39)
Cortex (n = 9)
Pituitary (n = 29)
Yields of simultaneously isolated RNA and DNA from decreasing numbers of Neuro2a cells
Cell number (n = 3)
Total DNA yield [μg]
Total RNA yield [μg]
31 ± 1
12,7 ± 0,4
13 ± 0,8
5,4 ± 0,13
6,7 ± 0,6
2,8 ± 0,05
3,3 ± 0,25
1,44 ± 0,14
1,5 ± 0,07
0,59 ± 0,05
0,81 ± 0,07
0,41 ± 0,02
0,46 ± 0,06
0,29 ± 0,03
0,2 ± 0,05
0,25 ± 0,013
For purification of the DNA the other half of the lysate is equilibrated with equal volumes of Buffer AL and 100% EtOH and loaded on a Spin Column (Qiagen, DNeasy Blood and Tissue Kit). After subsequent washing steps according to the manufacturer's protocol the DNA is eluted with 70°C warm Buffer AE after a 10 min pre-incubation at 70°C. All tissue punches yielded enough DNA (Table 2) for sodium bisulfite treatment using 100 ng of DNA with the Epitect bisulfite kit (Qiagen). Starting material of less than 5,000 Neuro2a cells did not yielded satisfactory quantities of DNA (Table 3).
Results and Discussion
DNA and RNA yields and absorbance rates for all methods studied are listed in Table 1. The most efficient method for brain tissue punches among the different DNA extraction protocols is the SDS/Proteinase K method with a total yield of 850 ng per punch followed by the CTAB method (550 ng) and the DNeasy and Puregene isolation Kits (both 360 ng). All methods resulted in A260/280 values between 1,8 and 2,0 indicating only minor protein contamination. For the tested RNA methods, the Chomczynski protocol yielded the highest amount of RNA per punch (838 ng) with an A260/280 ratio of 1,9, indicating rather pure RNA. The Trizol method resulted in slightly less RNA (690 ng) and low A260/280 values of 1,7 indicating some protein contamination. The MN Nucleospin columns gave the least amount of RNA (366 ng).
The newly developed simultaneous DNA and RNA extraction had the highest DNA recovery with 765 ng per punch considering that just half a punch is subjected to extraction. The RNA yield from the simultaneous extraction is comparable to the Trizol method, but with a higher purity as indicated by the A260/280 ratio of 1,9.
Low yields of DNA and RNA led to reduced 260/230 ratios for all methods tested, however, increasing the starting material improved this parameter (Table 3). Importantly, downstream applications such as PCR, RT-PCR, bisulfite treatment and subsequent bisulfite PCR are not compromised by lower 260/230 values, as shown below. Nevertheless, other downstream applications should be tested prior to sample processing.
In summary, we have refined and verified a streamlined protocol for the isolation of high qualities and quantities of DNA and RNA from small tissues for the study of DNA methylation and mRNA expression. Such a procedure will facilitate the analysis of the role of DNA methylation on gene expression through direct correlation analysis.
This work was funded by the European Union (CRESCENDO - EU Contract No. LSHM-CT-2005-018652 to O.F.X. Almeida and D. Spengler).
- Clark SJ, Harrison J, Paul CL, Frommer M: High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994, 22: 2990-7. 10.1093/nar/22.15.2990.PubMedPubMed CentralView ArticleGoogle Scholar
- Laird PW: Principles and challenges of genome-wide DNA methylation analysis. Nat Rev Genet. 2010, 11: 191-203.PubMedView ArticleGoogle Scholar
- Grunau C, Clark SJ, Rosenthal A: Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 2001, 29: 65-10.1093/nar/29.13.e65.View ArticleGoogle Scholar
- Chey S, Claus C, Liebert UG: Improved method for simultaneous isolation of proteins and nucleic acids. Anal Biochem. 2010, 411: 164-6.PubMedView ArticleGoogle Scholar
- Xu C, Houck JR, Fan W, Wang P, Chen Y, Upton M, Futran ND, Schwartz SM, Zhao LP, Chen C, Mendez E: Simultaneous isolation of DNA and RNA from the same cell population obtained by laser capture microdissection for genome and transcriptome profiling. J Mol Diagn. 2008, 10: 129-34. 10.2353/jmoldx.2008.070131.PubMedPubMed CentralView ArticleGoogle Scholar
- Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987, 162: 156-9.PubMedView ArticleGoogle Scholar
- Chomczynski P, Sacchi N: The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 2006, 1: 581-5. 10.1038/nprot.2006.83.PubMedView ArticleGoogle Scholar
- Lippke JA, Strzempko MN, Raia FF, Simon SL, French CK: Isolation of intact high-molecular-weight DNA by using guanidine isothiocyanate. Appl Environ Microbiol. 1987, 53: 2588-9.PubMedPubMed CentralGoogle Scholar
- Wright TL, Mamish D, Combs C, Kim M, Donegan E, Ferrell L, Lake J, Roberts J, Ascher NL: Hepatitis B virus and apparent fulminant non-A, non-B hepatitis. Lancet. 1992, 339: 952-5. 10.1016/0140-6736(92)91530-L.PubMedView ArticleGoogle Scholar
- de Tomaso AW, Weissman IL: Construction and characterization of large-insert genomic libraries (BAC and fosmid) from the Ascidian Botryllus schlosseri and initial physical mapping of a histocompatibility locus. Mar Biotechnol (NY). 2003, 5: 103-15.Google Scholar
- Kotlowski R, Martin A, Ablordey A, Chemlal K, Fonteyne PA, Portaels F: One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish. J Med Microbiol. 2004, 53: 927-33. 10.1099/jmm.0.45593-0.PubMedView ArticleGoogle Scholar
- Dyachenko V, Beck E, Pantchev N, Bauer C: Cost-effective method of DNA extraction from taeniid eggs. Parasitol Res. 2008, 102: 811-3. 10.1007/s00436-007-0855-6.PubMedView ArticleGoogle Scholar
- Coombs LM, Pigott D, Proctor A, Eydmann M, Denner J, Knowles MA: Simultaneous isolation of DNA. RNA, and antigenic protein exhibiting kinase activity from small tumor samples using guanidine isothiocyanate. Anal Biochem. 1990, 188: 338-43. 10.1016/0003-2697(90)90617-I.PubMedView ArticleGoogle Scholar
- Triant DA, Whitehead A: Simultaneous extraction of high-quality RNA and DNA from small tissue samples. J Hered. 2009, 100: 246-50.PubMedView ArticleGoogle Scholar
- Winnepenninckx B, Backeljau T, De Wachter R: Extraction of high molecular weight DNA from molluscs. Trends Genet. 1993, 9: 407-PubMedView ArticleGoogle Scholar
- Strauss WM: Preparation of genomic DNA from mammalian tissue. Current protocols in molecular biology. Edited by: Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. 1998, New York: John Wiley & Sons, 2: 2.2.1-2.2.3Google Scholar
- Varrault A, Bilanges B, Mackay DJ, Basyuk E, Ahr B, Fernandez C, Robinson DO, Bockaert J, Journot L: Characterization of the methylation-sensitive promoter of the imprinted ZAC gene supports its role in transient neonatal diabetes mellitus. J Biol Chem. 2001, 276: 18653-6. 10.1074/jbc.C100095200.PubMedView ArticleGoogle Scholar
- Murgatroyd C, Patchev AV, Wu Y, Micale V, Bockmühl Y, Fischer D, Holsboer F, Wotjak CT, Almeida OF, Spengler D: Dynamic DNA methylation programs persistent adverse effects of early-life stress. Nat Neurosci. 2009, 12: 1559-66. 10.1038/nn.2436.PubMedView ArticleGoogle Scholar
- Ikeda H, Osakada F, Watanabe K, Mizuseki K, Haraguchi T, Miyoshi H, Kamiya D, Honda Y, Sasai N, Yoshimura N, Takahashi M, Sasai Y: Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells. Proc Natl Acad Sci USA. 2005, 102: 11331-6. 10.1073/pnas.0500010102.PubMedPubMed CentralView ArticleGoogle Scholar
- Nettersheim D, Biermann K, Gillis AJ, Steger K, Looijenga LH, Schorle H: NANOG promoter methylation and expression correlation during normal and malignant human germ cell development. Epigenetics. 2001, 6: 114-22.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.