scholarly journals Optimizing preservation protocols to extract high‐quality RNA from different tissues of echinoderms for next‐generation sequencing

2013 ◽  
Vol 13 (5) ◽  
pp. 884-889 ◽  
Author(s):  
Rocío Pérez‐Portela ◽  
Ana Riesgo
Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1691
Author(s):  
Muscarella ◽  
Fabrizio ◽  
De Bonis ◽  
Mancini ◽  
Balsamo ◽  
...  

Thanks to personalized medicine trends and collaborations between industry, clinical research groups and regulatory agencies, next generation sequencing (NGS) is turning into a common practice faster than one could have originally expected. When considering clinical applications of NGS in oncology, a rapid workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, as well as producing high quality library preparation, can be real challenges. Here we consider these targets and how applying effective automation technology to NGS workflows may help improve yield, timing and quality-control. We firstly evaluated DNA recovery from archived FFPE blocks from three different manual extraction methods and two automated extraction workstations. The workflow was then implemented to somatic (lung/colon panel) and germline (BRCA1/2) library preparation for NGS analysis exploiting two automated workstations. All commercial kits gave good results in terms of DNA yield and quality. On the other hand, the automated workstation workflow has been proven to be a valid automatic extraction system to obtain high quality DNA suitable for NGS analysis (lung/colon Ampli-seq panel). Moreover, it can be efficiently integrated with an open liquid handling platform to provide high-quality libraries from germline DNA with more reproducibility and high coverage for targeted sequences in less time (BRCA1/2). The introduction of automation in routine workflow leads to an improvement of NGS standardization and increased scale up of sample preparations, reducing labor and timing, with optimization of reagents and management.


2019 ◽  
Vol 110 (8) ◽  
pp. 2652-2657 ◽  
Author(s):  
Hidenori Kage ◽  
Shinji Kohsaka ◽  
Aya Shinozaki‐Ushiku ◽  
Yoshihisa Hiraishi ◽  
Jiro Sato ◽  
...  

2015 ◽  
Vol 24 ◽  
pp. 115-118 ◽  
Author(s):  
Sneha Asai ◽  
Adrianna Ianora ◽  
Chiara Lauritano ◽  
Penelope K. Lindeque ◽  
Ylenia Carotenuto

Epigenetics ◽  
2013 ◽  
Vol 8 (5) ◽  
pp. 542-547 ◽  
Author(s):  
Karolina A. Aberg ◽  
Lin Y. Xie ◽  
Srilaxmi Nerella ◽  
William E. Copeland ◽  
E. Jane Costello ◽  
...  

2011 ◽  
Vol 22 (1) ◽  
pp. 125-133 ◽  
Author(s):  
N. J. Parkinson ◽  
S. Maslau ◽  
B. Ferneyhough ◽  
G. Zhang ◽  
L. Gregory ◽  
...  

2020 ◽  
Author(s):  
Buddhini Ranawaka ◽  
Milos Tanurdzic ◽  
Peter Waterhouse ◽  
Fatima Naim

Abstract Background All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the differences in chromatin landscape of this unique collection will shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. N.benthamiana is also an important biotechnological tool and it is widely used in virological research and functional genomics. Chromatin Immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is well established technique used to study histone modifications. However, due to the starchy nature of mature N.benthamiana leaves, previously published protocols were unsuitable. The aim of this study was to optimise ChIP protocol for N.benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Results Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. The higher amounts of starch in mature N.benthamiana leaves that co-precipitated with nuclei using previously published protocols, hindered chromatin shearing and resulted in low recovery of ChIP DNA. The optimised method reduced starch contamination and resulted in isolation of high quality nuclei suitable for next generation sequencing. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Conclusions An optimised ChIP method for mature leaves of N.benthamiana is described. It is relatively less laborious than previously published protocols and allows extraction of high quality nuclear genomic DNA from N.benthamiana. It is also the first comprehensive ChIP method for starchy leaves of N.benthamiana suitable for preparation of ChIP libraries for next generation sequencing.


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