RT-qPCR profiling of the rRNA genes <i>RN18S</i> and <i>RN28S</i> (A) and assessment of RNA integrity (B).

<p>RT-qPCR profiling of the rRNA genes <i>RN18S</i> and <i>RN28S</i> (A) and assessment of RNA integrity (B).</p

Processing of RNA for expression uniformity analysis by RT-qPCR.

<p>*Input RNA might contain nucleotides or short DNA fragments as a result of DNase digestion of co-purified DNA. The same amount of RNA was used for RT as concluded from a second spectrophotometric measurement of RNA mass before the RT step and based on the subsequent determination of <i>Cq</i> values for the rRNA genes <i>RN18S</i> and <i>RN28S</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142122#pone.0142122.g002" target="_blank">Fig 2</a>).</p

Details on qPCR assays.

<p>F and R: forward and reverse primers; <i>E</i>: amplification efficiency; NA: information not available due to incomplete sequence data.</p><p>Hs: <i>Homo sapiens</i>.</p><p>Assay ID according to RTPrimerDB (<a href="http://medgen.ugent.be/rtprimerdb/" target="_blank">http://medgen.ugent.be/rtprimerdb/</a>).</p><p>Details on qPCR assays.</p

Expression uniformity analysis using the RefFinder software.

<p>The individual genes or the normalization factor calculated from the best-ranking, not co-expressed pair of genes are depicted. Only the “expressors” of highest and lowest ranks are presented.</p

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer-2

Tings from an alignment of concatenated mtDNA sequences (3806 nucleotides: control region, , , and ) of the three donor cell lines, their SCNT clones (CA1 to CA7, CB1 to CB3, and CC1 and CC2) and of an European mitochondrial reference genome (GenBank:). The scale bar represents 0.001 substitutions per site, and quartet puzzling values are shown (all are >50). The numbers at the nodes (quartet puzzling values) indicate the frequencies of occurrence for 1,000 replicate trees. Quartet puzzling support values provide an estimate of support of a given branch and can be interpreted in much the same way as bootstrap values. CA5 is the most divergent donor A-derived clone which is highlighted by the ellipse (see below for amino acid changes).<p><b>Copyright information:</b></p><p>Taken from "Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer"</p><p>http://www.biomedcentral.com/1471-213X/7/141</p><p>BMC Developmental Biology 2007;7():141-141.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2323970.</p><p></p

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer-0

Tings from an alignment of concatenated mtDNA sequences (3806 nucleotides: control region, , , and ) of the three donor cell lines, their SCNT clones (CA1 to CA7, CB1 to CB3, and CC1 and CC2) and of an European mitochondrial reference genome (GenBank:). The scale bar represents 0.001 substitutions per site, and quartet puzzling values are shown (all are >50). The numbers at the nodes (quartet puzzling values) indicate the frequencies of occurrence for 1,000 replicate trees. Quartet puzzling support values provide an estimate of support of a given branch and can be interpreted in much the same way as bootstrap values. CA5 is the most divergent donor A-derived clone which is highlighted by the ellipse (see below for amino acid changes).<p><b>Copyright information:</b></p><p>Taken from "Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer"</p><p>http://www.biomedcentral.com/1471-213X/7/141</p><p>BMC Developmental Biology 2007;7():141-141.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2323970.</p><p></p

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer-3

-oocyte mtDNA) and ARMS-qPCR (non-discriminative (ND) and discriminative (D) assays) for the low-abundant SNP variant (target here: donor mtDNA) at the mutated site. The amplification efficiency of the D assay was 91%. Without further optimization concerning conditions for enzymatic digestion, quantification by REMS-qPCR targeting a single donor-B-specific SNP reached a detection limit of 0.02%, i.e. a point mutation discrimination selectivity factor of 5 × 10. It allowed detection of heteroplasmy (0.1%) in the donor B-derived clone CB2. This could not be detected by conventional ARMS-qPCR discriminating point mutations only down to 0.1% (see: illustrated improvement of discrimination). For clarity each plot is presented as the mean calculated from duplicate amplification reactions. Individual Ct values, i.e. PCR cycle numbers at which plots crossed an arbitrarily placed signal threshold, are given in the figure key. An independent technical replicate of this REMS-qPCR experiment demonstrated reproducibility of the method (data not shown).<p><b>Copyright information:</b></p><p>Taken from "Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer"</p><p>http://www.biomedcentral.com/1471-213X/7/141</p><p>BMC Developmental Biology 2007;7():141-141.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2323970.</p><p></p

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer-1

-oocyte mtDNA) and ARMS-qPCR (non-discriminative (ND) and discriminative (D) assays) for the low-abundant SNP variant (target here: donor mtDNA) at the mutated site. The amplification efficiency of the D assay was 91%. Without further optimization concerning conditions for enzymatic digestion, quantification by REMS-qPCR targeting a single donor-B-specific SNP reached a detection limit of 0.02%, i.e. a point mutation discrimination selectivity factor of 5 × 10. It allowed detection of heteroplasmy (0.1%) in the donor B-derived clone CB2. This could not be detected by conventional ARMS-qPCR discriminating point mutations only down to 0.1% (see: illustrated improvement of discrimination). For clarity each plot is presented as the mean calculated from duplicate amplification reactions. Individual Ct values, i.e. PCR cycle numbers at which plots crossed an arbitrarily placed signal threshold, are given in the figure key. An independent technical replicate of this REMS-qPCR experiment demonstrated reproducibility of the method (data not shown).<p><b>Copyright information:</b></p><p>Taken from "Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer"</p><p>http://www.biomedcentral.com/1471-213X/7/141</p><p>BMC Developmental Biology 2007;7():141-141.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2323970.</p><p></p

DataSheet1_Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma.zip

We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%–65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%–95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18–55 years). In general, inter-individual variance of miRNA abundance was low or very low as indicated by coefficient of variation (CV) values of 0.6%–8.2%. miR-3665 and miR-1915-3p outperformed in this analysis (CVs: 0.6 and 2.4%, respectively). The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes.</p

Supplemental Material - Cellular and Site-Specific Mitochondrial Characterization of Vital Human Amniotic Membrane

<p>Supplemental Material for Cellular and Site-Specific Mitochondrial Characterization of Vital Human Amniotic Membrane by Asmita Banerjee, Andrea Lindenmair, Simone Hennerbichler, Philipp Steindorf, Ralf Steinborn, Andrey V. Kozlov, Heinz Redl, Susanne Wolbank, and Adelheid Weidinger in Cell Transplantation</p