Small RNA-Seq Library Prep Kit Publications
Small RNA-Seq Publications
Dr. Katia Baruffi
About one third of patients with epilepsy are pharmacoresistant and suffer from poor quality of life, seizure-related accidents and comorbidities, increased risk of death and disabling psychosocial consequences. Clinical outcomes for patients unresponsive to current pharmacological tools could be improved by characterization and development of biomarkers that could assist physicians in either (i) optimizing response to treatment with available antiepileptic drugs or (ii) identifying early those patients who are …
Features Small RNA-Seq Library Prep Kit for Illumina
Fatima Heinicke, Xiangfu Zhong, Manuela Zucknick, Johannes Breidenbach, Arvind Y. M. Sundaram, Siri T. Flåm, Magnus Leithaug, Marianne Dalland, Andrew Farmer, Jordana M. Henderson, Melanie A. Hussong, Pamela Moll, Loan Nguyen, Amanda McNulty, Jonathan M. Shaffer, Sabrina Shore, Hoichong Karen Yip, Jana Vitkovska, Simon Rayner, Benedicte A Lie & Gregor D. Gilfillan
High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.
Melanie Villanueva, Connor Michie, Sandrine Parent, Georges N Kanaan, Ghazaleh Rafatian, Pushpinder Kanda, Bin Ye, Wenbin Liang, Mary-Ellen Harper, Darryl R Davis
Decades of work have shown that diabetes increases the risk of heart disease and worsens clinical outcomes after myocardial infarction. Because diabetes is an absolute contraindication to heart transplant, cell therapy is increasingly being explored as a means of improving heart function for these patients with very few other options. Given that hyperglycemia promotes the generation of toxic metabolites, the influence of the key detoxification enzyme glyoxalase 1 (Glo1) on chronic hyperglycemia induced heart explant-derived cell (EDC) dysfunction was investigated.
Methods: EDCs were cultured from wild type C57Bl/6 or Glo1 over-expressing transgenic mice 2 months after treatment with the pancreatic beta cell toxin streptozotocin or vehicle. The effects of Glo1 overexpression was evaluated using in vitro and in vivo models of myocardial ischemia.
Results: Chronic hyperglycemia reduced overall culture yields and increased the reactive dicarbonyl cell burden within EDCs. These intrinsic cell changes reduced the angiogenic potential and production of pro-healing exosomes while promoting senescence and slowing proliferation. Compared to intra-myocardial injection of normoglycemic cells, chronic hyperglycemia attenuated cell-mediated improvements in myocardial function and reduced the ability of transplanted cells to promote new blood vessel and cardiomyocyte growth. In contrast, Glo1 overexpression decreased oxidative damage while restoring both cell culture yields and EDC-mediated repair of ischemic myocardium. The latter was associated with enhanced production of pro-healing extracellular vesicles by Glo1 cells without altering the pro-healing microRNA cargo within.
Conclusions: Chronic hyperglycemia decreases the regenerative performance of EDCs. Overexpression of Glo1 reduces dicarbonyl stress and prevents chronic hyperglycemia-induced dysfunction by rejuvenating the production of pro-healing extracellular vesicles.
Keywords: cardiac stem cells, diabetes, extracellular vesicles, hyperglycemia, heart failure, myocardial infarction, oxidative stress, reactive dicarbonyls
Iryna Lytvynenko, Helge Paternoga, Anna Thrun, Annika Balke, Tina A. Müller, Christina H. Chiang, Katja Nagler, George Tsaprailis, Simon Anders, Ilka Bischofs, Julie A. Maupin-Furlow, Christian M.T. Spahn, Claudio A.P. Joazeiro
In ribosome-associated quality control (RQC), Rqc2/NEMF closely supports the E3 ligase Ltn1/listerin in promoting ubiquitylation and degradation of aberrant nascent-chains obstructing large (60S) ribosomal subunits—products of ribosome stalling during translation. However, while Ltn1 is eukaryote-specific, Rqc2 homologs are also found in bacteria and archaea; whether prokaryotic Rqc2 has an RQC-related function has remained unknown. Here, we show that, as in eukaryotes, a bacterial Rqc2 homolog (RqcH) recognizes obstructed 50S subunits and promotes nascent-chain proteolysis. Unexpectedly, RqcH marks nascent-chains for degradation in a direct manner, by appending C-terminal poly-alanine tails that act as degrons recognized by the ClpXP protease. Furthermore, RqcH acts redundantly with tmRNA/ ssrA and protects cells against translational and environmental stresses. Our results uncover a proteolytic-tagging mechanism with implications toward the function of related modifications in eukaryotes and suggest that RQC was already active in the last universal common ancestor (LUCA) to help cope with incomplete translation.
Ryan M. Sheridan, Nova Fong, Angelo D’Alessandro, David L. Bentley
In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. How TFIIS affects transcription under normal conditions is poorly understood. We identified backtracking sites in human cells using a dominant-negative TFIIS (TFIIS DN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3′ flanking regions where termination is enhanced by TFIIS DN, suggesting that backtracked pol II is a favorable substrate for termination. Rescue from backtracking by RNA cleavage also promotes escape from 5′ pause sites, prevents premature termination of long transcripts, and enhances activation of stress-inducible genes. TFIIS DN slowed elongation rates genome-wide by half, suggesting that rescue of backtracked pol II by TFIIS is a major stimulus of elongation under normal conditions.