High-quality RNA and DNA from flow cytometrically sorted human epithelial cells and tissues

Microarray technologies have made possible comprehensive analyses of nucleic acid sequence and expression. However, the technology to obtain efficiently high-quality RNA and DNA suitable for array analysis from purified populations of neoplastic cells from human tissues has not been well addressed. Microdissection can enrich for populations of cells present in various tumor tissues, but it is not easily automated or performed rapidly, and there are tissues in which cells of interest cannot be readily isolated based on morphologic criteria alone. Here we describe a protocol for efficient RNA and DNA isolation from flow cytometrically purified whole epithelial cells from primary tissue. The aqueous reagent, RNAlater, which preserves RNA, allows immunolabeling and purification of whole epithelial cells by flow sorting without special instrument preparation to reduce RNase activity. We used real-time PCR to determine RNA quality afterflow sorting. High-quality RNA and DNA suitable for expression and genotype analysis can be readily obtained from flow cytometrically purified populations of neoplastic cells from human tissues.     

Purification of DNA from formaldehyde fixed and paraffin embedded human tissue

The ability to isolate DNA from preserved human tissues would provide numerous experimental opportunities. In this report it is shown that DNA can be extracted from tissues prepared for routine histopathological examination (i.e., fixed with formaldehyde and embedded in paraffin). Although the extracted DNA is not intact, it is double stranded, cleavable with restriction endonucleases, and suitable for a variety of standard techniques used in molecular biology.     

Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells.

Analysis of the total base composition of DNA from seven different normal human tissues and eight different types of homogeneous human cell populations revealed considerable tissue-specific and cell-specific differences in the extent of methylation of cytosine residues. The two most highly methylated DNAs were from thymus and brain with 1.00 and 0.98 mole percent 5-methylcytosine (m5C), respectively. The two least methylated DNAs from in vivo sources were placental DNA and sperm DNA, which had 0.76 and 0.84 mole percent m5C, respectively. The differences between these two groups of samples were significant with p less than 0.01. The m5C content of DNA from six human cell lines or strains ranged from 0.57 to 0.85 mole percent. The major and minor base composition of DNA fractionated by reassociation kinetics was also determined. The distribution of m5C among these fractions showed little or no variation with tissue or cell type with the possible exception of sperm DNA. In each case, nonrepetitive DNA sequences were hypomethylated compared to unfractionated DNA.     

32P-postlabelling analysis of DNA from human tissues.

DNA extracted from human lung, bladder, liver, pancreas, cervix and breast tissue samples taken at autopsy (37 sample sets) was analysed by the nuclease P1 enhancement modification of the 32P-postlabelling assay for levels of aromatic carcinogen DNA adducts. Results were combined with those from a previous study for statistical analysis of 56 sample sets (32 male+24 female). A strong trend was seen for increased adduct levels in the lung DNA of smokers and a weak association for the bladder DNA of smokers compared to non-smokers. Aromatic adducts were also detected in other tissues.     

DNA damage response and cellular senescence in tissues of aging mice

The impact of cellular senescence onto aging of organisms is not fully clear, not at least because of the scarcity of reliable data on the mere frequency of senescent cells in aging tissues. Activation of a DNA damage response including formation of DNA damage foci containing activated H2A.X (γ-H2A.X) at either uncapped telomeres or persistent DNA strand breaks is the major trigger of cell senescence. Therefore, γ-H2A.X immunohistochemistry (IHC) was established by us as a reliable quantitative indicator of senescence in fibroblasts in vitro and in hepatocytes in vivo and the age dependency of DNA damage foci accumulation in ten organs of C57Bl6 mice was analysed over an age range from 12 to 42 months. There were significant increases with age in the frequency of foci-containing cells in lung, spleen, dermis, liver and gut epithelium. In liver, foci-positive cells were preferentially found in the centrilobular area, which is exposed to higher levels of oxidative stress. Foci formation in the intestine was restricted to the crypts. It was not associated with either apoptosis or hyperproliferation. That telomeres shortened with age in both crypt and villus enterocytes, but telomeres in the crypt epithelium were longer than those in villi at all ages were confirmed by us. Still, there was no more than random co-localization between γ-H2A.X foci and telomeres even in crypts from very old mice, indicating that senescence in the crypt enterocytes is telomere independent. The results suggest that stress-dependent cell senescence could play a causal role for aging of mice.     

Extraction of DNA from milligram amounts of fresh,herbarium and mummified plant tissues

Summary We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3–200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22–118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.     

Nucleotide sequence identity of mitochondrial DNA from different human tissues

Recombinant DNA techniques have been used to search for mitochondrial (mt) nucleotide (nt) sequence differences between human tissues within an individual. mtDNA isolated from brain, heart, liver, kidney, and skeletal muscle of two different individuals was cleaved with SacI and XbaI, and then cloned in bacteriophage M13. Partial nt sequence determination of 121 independently isolated recombinant M13 clones containing either the cytochrome oxidase subunit III gene or the D-loop region of human mtDNA revealed base substitution differences between individuals, and between each individual and the published human mtDNA sequence. A majority of these base substitutions were transitions. No systematic nt sequence differences were identified between tissues within an individual, however. These results suggest that mtDNA sequence alterations do not accompany organogenesis, and that somatic mutations do not accumulate in the mtDNA of different human tissues to a level of greater than one nt substitution per molecule.     

Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues

Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns of WGBS, and develop and assess the accuracy of a new protocol for mtDNA nucleotide variant-specific methylation using single-molecule Oxford Nanopore Sequencing (ONS). Our approach circumvents confounders by enriching for full-length molecules over nuclear DNA. Variant calling analysis against showed that 99.5% of homoplasmic mtDNA variants can be reliably identified providing there is adequate sequencing depth. We show that some of the mtDNA methylation signal detected by ONS is due to sequence-specific false positives introduced by the technique. The residual signal was observed across several human primary and cancer cell lines and multiple human tissues, but was always below the error threshold modelled using negative controls. We conclude that there is no evidence for CpG methylation in human mtDNA, thus resolving previous controversies. Additionally, we developed a reliable protocol to study epigenetic modifications of mtDNA at single-molecule and single-base resolution, with potential applications beyond CpG methylation.     

Biosynthesis and cellular localization of embryonal prealbumin in human embryonal tissues

Immunofluorescent analysis of chorion sections and various embryonal tissues revealed localization of embryonal prealbumin (EPA) in the mesenchymal cells of the chorion, bones, umbilical cord, skin and in the cytoplasm of the epithelium distal part of the embryonal kidney canals. Synthesis of EPA slow peak in the suspension tissue culture of the chorion, umbilical cord and bones was shown. It is suggested that EPA is a mesenchyme product which is actively synthesized during the period of embryonal development. EPA is resynthesized and localized in the cells of tumors originated from the connective tissue. Cells of the tumors of non-connective origin did not contain EPA, whereas it was detected in the cells of the adjacent connective tissue. The phenomenon of embryonal reversion, probably, takes place not only in the "original" tumor cells but also in the surrounding connective tissues.     

A fixed site of DNA replication in eucaryotic cells

We studied the role of the nuclear matrix (the skeletal framework of the nucleus) in DNA replication both in vivo and in a cell culture system. When regenerating rat liver or exponentially growing 3T3 fibroblasts are pulse-labeled with ³H-thymidine and nuclear matrix is subsequently isolated, the fraction of DNA remaining tightly attached to the matrix is highly enriched in newly synthesized DNA. After a 30 sec pulse labeling period and limited DNAase I digestion, the matrix DNA of 3T3 fibroblasts, which constitutes 15% of the total DNA, contains approximately 90% of the labeled newly synthesized DNA. Over 80% of this label can be chased out of the matrix DNA if the pulse is followed by a 45 min incubation with excess unlabeled thymidine. These and other kinetic studies suggest that the growing point of DNA replication is attached to the nuclear matrix. Studies measuring the size distribution of the matrix DNA also support this conclusion. Reconstitution controls and autoradiographic studies indicate that these results are not due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. Electron microscopic autoradiography shows that, as with intact nuclei, sites of DNA replication are distributed throughout the nuclear matrix. A fixed site of DNA synthesis is proposed in which DNA replication complexes are anchored to the nuclear matrix and the DNA is reeled through these complexes as it is replicated.     

Methylation sequencing from limiting DNA: embryonic,fixed, and microdissected cells

It is frequently useful to determine the methylation state of samples containing limited amounts of DNA such as from embryos, or from fixed tissue samples in which DNA is degraded or difficult to isolate. By modification of the standard protocols for DNA preparation and bisulfite treatment, it is possible to obtain DNA methylation sequence data for such samples. We present methods for bisulfite treatment of embryos, fixed sections, and samples obtained by laser capture microdissection, and discuss the additional experimental considerations required when working with small numbers of cells or degraded DNA samples.