Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance

Ford, K L; Anwar, Maryam; Heys, Rachael; Ahmed, Eltayeb Mohamed; Caputo, Massimo; Gamé, Laurence; Reeves, Barnaby C; Punjabi, Prakash P; Angelini, Gianni; Petretto, Enrico; Emanueli, Costanza

doi:10.1371/journal.pone.0213685

Cited by 14 publications

(13 citation statements)

References 49 publications

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“…. The variability observed in RNA isolation yields with commercial kits is in agreement with previous observations 56,57 . We further add glycogen during isopropanol precipitation to help recover all RNA.…”

supporting

confidence: 91%

Dissection of <em>Drosophila melanogaster</em> Flight Muscles for Omics Approaches

Kao¹,

Nikonova²,

Ravichandran³

et al. 2019

JoVE

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Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (<48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (>48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis. Video Link The video component of this article can be found at https://www.jove.com/video/60309/ Drosophila melanogaster is another well-established genetic model organism. The structure of the sarcomere as well as individual sarcomere components are highly conserved from flies to vertebrates 4,9,10 , and the indirect flight muscles (IFMs) have become a powerful model to study multiple aspects of muscle development 11,12. First, the fibrillar flight muscles are functionally and morphologically distinct from tubular body muscles 11,13 , allowing investigation of muscle-type specific developmental mechanisms. Transcription factors including Spalt major (Salm) 14 , Extradenticle (Exd), and Homothorax (Hth) 15 have been identified as fibrillar fate regulators. Additionally, downstream of Salm, the CELF1 homolog Bruno1 (Bru1, Aret) directs a fibrillar-specific splicing program 16,17. Second, IFMs are an important model for understanding the process of myogenesis itself, from myoblast fusion and myotube attachment to myofibrillogenesis and sarcomere maturation 9,18,19. Third, Drosophila genetics permits investigation of contributions by individual proteins, protein domains, and protein isoforms to sarcomere formation, function, and biophysical properties 20,21,22,23. Lastly, IFM models have been developed for the study of...

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supporting

confidence: 91%

Dissection of <em>Drosophila melanogaster</em> Flight Muscles for Omics Approaches

Kao¹,

Nikonova²,

Ravichandran³

et al. 2019

JoVE

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“…The unsuccessful optimisation of the standard Qiagen mIRNeasy protocol is in contrast with previously published literature using human plasma samples, suggesting that miRNA recovery was improved by carriers such as glycogen ( 50 ) or yeast ( 42 , 51 ). However, one of these studies used pig instead of human plasma for the kit optimisation experiments, failing to re-test the findings upon human samples; a precarious assumption given the known variability in media and kit performance between different species and disease states ( 25 , 35 ).…”

Section: Discussioncontrasting

confidence: 68%

Comparison and optimisation of microRNA extraction from the plasma of healthy pregnant women

et al. 2021

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circulating microrna (mirna) biomarkers are implicated in the diagnosis, monitoring and prediction of various disease processes. Before embarking upon biomarker discovery, miRNA extraction techniques must first be optimised in the biofluid and population under study. Using plasma from a healthy pregnant woman, it was attempted to optimise and compare the performance of two commercially available mirna extraction kits; Qiagen (mirneasy Serum/Plasma) and Promega (Maxwell ® rSc mirna from Tissue or Plasma or Serum). Sample mirna content (concentration and percentage) was assessed using agilent Bioanalyzer Small rna chips and reverse transcription-quantitative Pcr (rT-qPcr) using four constitutively expressed mirnas (hsa-mir-222-3p, hsa-let-7i-3p, hsa-mir-148-3p and hsa-mir-30e-5p). Quality control spike-ins monitored rna extraction (uniSp2, 4 and 5) and cdna synthesis (uniSp6, cel-mir-39-3p) efficiency. optimisation approaches included: i) Starting volume of plasma; the addition of ii) Proteinase K; iii) a rna bacteriophage carrier (MS2); and iv) a glycogen carrier. The two kits exhibited equivalence in terms of mirna recovery based on Bioanalyzer and rT-qPcr ΔΔcq results. optimisation attempts for both kits failed to improve upon mirna content compared with standard methodology. comparing the standard methodology, the Qiagen kit was more consistent (smaller variance of Δcq values) compared with the Promega kit. The standard methodology of either kit would be suitable for the investigation of mirna biomarkers in a healthy pregnant population.

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“…Comparison of commercial miRNA isolation kits based on operational factors. in isolation of selective miRNA populations, as well as often resulting in lower quantities of impure total RNA from smaller sample volumes 14,20,21 . A similar study comparing isolation kits miRNA recovery from both plasma and cerebrospinal fluid showed that TRIzol did not perform as well as commercial extraction kits, even when combined with on-column clean-up 22 .…”

Section: Discussionmentioning

confidence: 99%

Comparison of methods for miRNA isolation and quantification from ovine plasma

Wright

Silva

Purdie

et al. 2020

Sci Rep

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c. purdie & Karren M. plain microRNA (miRNA) are promising candidates for disease biomarkers as they are abundant in circulation, highly stable in biological fluids and may yield diagnostic biomarker signatures. The reported issues with miRNA isolation using traditional RNA reagents necessitates the optimisation of miRNA isolation from challenging samples. In this study we compared six commercial RNA extraction kits to evaluate their ability to isolate miRNA from ovine plasma. We also compared three methods for quantification of small RNA extracted from plasma to determine the most reliable. Using minimal sample inputs of fresh and frozen plasma from five sheep, we compared the six kits (Kit A-F) using quantitative PCR. Operational factors were also assessed for each kit. Kits A and B provided the best detection of the miRNA qPCR reference genes across fresh and frozen samples (p < 0.001) followed by Kit C. The Qubit and microRNA assay provided the least variation (% CV 5.47, SEM ± 0.07), followed by the NanoDrop (% CV 7.01, SEM ± 0.92) and Agilent Bioanalyzer (% CV 59.21, SEM ± 1.31). We identify Kit A to be optimal for isolating miRNA from small volumes of fresh and frozen ovine plasma, and Kit B the top performing kit taking into consideration miRNA detection and operational factors. The Qubit fluorometer using a microRNA assay was the most reliable miRNA quantification method.

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Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance

Cited by 14 publications

References 49 publications

Dissection of <em>Drosophila melanogaster</em> Flight Muscles for Omics Approaches

Dissection of <em>Drosophila melanogaster</em> Flight Muscles for Omics Approaches

Comparison and optimisation of microRNA extraction from the plasma of healthy pregnant women

Comparison of methods for miRNA isolation and quantification from ovine plasma

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