UltraPrep is an open-source, two-step method for purification of cell-free DNA that entails extraction of total DNA followed by size-selective enrichment of the smaller fragments that are ...characteristic of DNA originating from fragmentation between nucleosome. The advantages of the two related protocols that are described are that they can easily accommodate a wide range of sample input volumes, they rely on simple, magnetic bead-based technology, the yields of cfDNA are directly comparable to the most popular methods for cfDNA purification, and they dramatically reduce the cost of cfDNA isolation relative to currently available commercial methods. We provide a framework for physical and molecular quality analysis of purified cfDNA and demonstrate that the cfDNA generated by UltraPrep meets or exceeds the quality metrics of the most commonly used procedure. In addition, our method removes high molecular weight genomic DNA (hmwgDNA) that can interfere with downstream assay results, thereby addressing one of the primary concerns for preanalytical collection of blood samples.
Genomic analysis of circulating, cell-free DNA (cfDNA) is being used extensively for molecular diagnostics. Many approaches rely on the construction of cfDNA genomic libraries, targeted retrieval of ...specific genomic regions and analysis by next-generation DNA sequencing. Several steps during sample preparation require isolation of DNA fragments within a particular size range. In this Benchmark article, two related methods for size-selective DNA fragment enrichment are described.
Two strategies for performing sequential, bead-based purification of DNA fragments are described. One procedure can be used to remove larger-than-desired DNA fragments while the other is designed to eliminate smaller-than-desired fragment ‘impurities’. Both methods achieve improved purification and size discrimination relative to current bead-based purification protocols.
The development of solid electrolytes with the combination of high ionic conductivity, electrochemical stability, and resistance to Li dendrites continues to be a challenge. A promising approach is ...to create inorganic–organic composites, where multiple components provide the needed properties, but the high sintering temperature of materials such as ceramics precludes close integration or co‐sintering. Here, new ceramic–salt composite electrolytes that are cold sintered at 130 °C are demonstrated. As a model system, composites of Li1.5Al0.5Ge1.5(PO4)3 (LAGP) or Li1+x
+y
Alx
Ti2−x
Siy
P3−y
O12 (LATP) with bis(trifluoromethanesulfonyl)imide (LiTFSI) salts are cold sintered. The resulting LAGP–LiTFSI and LATP–LiTFSI composites exhibit high relative densities of about 90% and ionic conductivities in excess of 10−4 S cm−1 at 20 °C, which are comparable with the values obtained from LAGP and LATP sintered above 800 °C. It is also demonstrated that cold sintered LAGP–LiTFSI is electrochemically stable in Li symmetric cells over 1800 h at 0.2 mAh cm−2. Cold sintering provides a new approach for bridging the gap in processing temperatures of different materials, thereby enabling high‐performance composites for electrochemical systems.
Ceramic–salt composite solid electrolytes are fabricated through cold sintering at 130 °C. Cold sintering enables integration of bis(trifluoromethanesulfonyl)imide (LiTFSI) with ceramics to achieve ionic conductivities near 10−4 S cm−1 and relative densities of ≈90%. Stable cycling over 1800 h in Li metal symmetric cells is also demonstrated.
The breadth of diagnostic procedures that utilize cell free DNA (cfDNA) from human plasma has increased dramatically in recent years. Here, we confirm that tumor-derived cfDNA fragments are similar ...in size distribution to cfDNA derived from normal tissues. Therefore, collection procedures optimized with healthy donor specimens are likely to be applicable to the diagnosis and monitoring of many different cancer types. We verify that the distribution and DNA sequences of fragmentation sites in cfDNA from both normal-germline and tumor-derived cfDNA are non-random. A broad survey of cfDNA from healthy donors suggests that average individuals possess ~6 ng of cfDNA per mL of plasma. Importantly, the cfDNA present in plasma samples that were initially collected as whole blood in K2-EDTA tubes and subsequently processed by centrifugation is stable for several days at ambient temperatures. This observation has the potential to significantly reduce the cost and logistical complexity of shipping clinical samples from the site of collection to centers proficient in diagnostic analysis. Finally, plasma samples collected with high-volume plasma collection devices possess abundant quantities of cfDNA. Since the quantity of analyzed cfDNA is directly proportional to sensitivity of diagnostic assays, this method of plasma collection, where available, could enable highly sensitive post-treatment disease monitoring and early detection of cancer in at-risk individuals.
The Colorectal MicroRNAome Cummins, Jordan M.; He, Yiping; Leary, Rebecca J. ...
Proceedings of the National Academy of Sciences - PNAS,
03/2006, Letnik:
103, Številka:
10
Journal Article
Recenzirano
Odprti dostop
MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have ...been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.
Studies of the host response to virus infection typically focus on protein-coding genes. However, non-protein-coding RNAs (ncRNAs) are transcribed in mammalian cells, and the roles of many of these ...ncRNAs remain enigmas. Using next-generation sequencing, we performed a whole-transcriptome analysis of the host response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection across four founder mouse strains of the Collaborative Cross. We observed differential expression of approximately 500 annotated, long ncRNAs and 1,000 nonannotated genomic regions during infection. Moreover, studies of a subset of these ncRNAs and genomic regions showed the following. (i) Most were similarly regulated in response to influenza virus infection. (ii) They had distinctive kinetic expression profiles in type I interferon receptor and STAT1 knockout mice during SARS-CoV infection, including unique signatures of ncRNA expression associated with lethal infection. (iii) Over 40% were similarly regulated in vitro in response to both influenza virus infection and interferon treatment. These findings represent the first discovery of the widespread differential expression of long ncRNAs in response to virus infection and suggest that ncRNAs are involved in regulating the host response, including innate immunity. At the same time, virus infection models provide a unique platform for studying the biology and regulation of ncRNAs.
There has been a surge of interest in the biology of microRNAs and the technology of RNA interference. We describe a simple, robust, inexpensive assay for quantitative analysis of microRNAs and ...short-interfering RNAs. The method relies on primer extension conversion of RNA to cDNA by reverse transcription followed by quantitative, real-time PCR. Technical parameters critical to the success of the assay are presented. Measurements of microRNA levels are sensitive, with most assays allowing measurements in the femtomolar range, which corresponds to tens of copies per cell or less. The assay has a high dynamic range and provides linear readout over differences in microRNA concentrations that span 6-7 orders of magnitude. The assay is capable of discriminating between related microRNA family members that differ by subtle sequence differences. We used the method for quantitative analysis of six microRNAs across 12 tissue samples. The data confirm striking variation in the patterns of expression of these noncoding regulatory RNAs.
We developed a procedure for the preparation of whole transcriptome cDNA libraries depleted of ribosomal RNA from only 1 μg of total RNA. The method relies on a collection of short, computationally ...selected oligonucleotides, called 'not-so-random' (NSR) primers, to obtain full-length, strand-specific representation of nonribosomal RNA transcripts. In this study we validated the technique by profiling human whole brain and universal human reference RNA using ultra-high-throughput sequencing.
Non-coding RNAs (ncRNAs) are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a ...polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6) is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I) cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available.
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