Human adipose and skeletal muscle tissue DNA, RNA, and protein content

Stroh, Andrew M.; Lynch, Colleen E.; Lester, Bridget E.; Minchev, Kiril; Chambers, Toby L.; Montenegro, Cristhian F.; Martinez, Clarisa Chavez; Fountain, William A.; Trappe, Todd A.; Trappe, Scott

doi:10.1152/japplphysiol.00343.2021

Cited by 12 publications

(6 citation statements)

References 63 publications

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“…Other critical variables also influenced the quality of the RNA, namely, the condition of the tissue or the impact of degradation during processing steps. The vulnerability of RNA in rat ILMs is in contrast to findings that characterize skeletal muscle mRNA as highly stable relative to the liver, brain, and adipose tissue [12][13][14] . This suggested that variable RNA stability across species, tissue types, and organ layers may require modified extraction strategies according to the specimen and sample size.…”

contrasting

confidence: 63%

“…integrity restricted the downstream assessment to a low-throughout application and perturbed the RT-qPCR performance. In contrast, living tissue samples following human muscle biopsy yielded between 456 to 676 ng total RNA and 7.9 ± 0.6 RIN 12 . Unfortunately, compared to other soft tissue types, the distinctive ILMs have not been amenable to RNA-Seq due to differences in cell characteristics 14 .…”

mentioning

confidence: 94%

“…Previous whole-genome analyses in the rat ILMs and other skeletal muscles utilized spin-column technology or reagents to separate the nucleic acids. Hence, similar protocols were evaluated in this investigation 12,[23][24][25]30 . Other methodological strategies for RNA purification, such as nucleic acid extraction via magnetic beads, were not applied here due to a lack of evidence of its use in rat skeletal muscles.…”

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confidence: 99%

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An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model

Kemfack

Hernández-Morato

Moayedi

et al. 2022

Sci Rep

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Challenges related to high-quality RNA extraction from post-mortem tissue have limited RNA-sequencing (RNA-seq) application in certain skeletal muscle groups, including the intrinsic laryngeal muscles (ILMs). The present study identified critical factors contributing to substandard RNA extraction from the ILMs and established a suitable method that permitted high-throughput analysis. Here, standard techniques for tissue processing were adapted, and an effective means to control confounding effects during specimen preparation was determined. The experimental procedure consistently provided sufficient intact total RNA (N = 68) and RIN ranging between 7.0 and 8.6, which was unprecedented using standard RNA purification protocols. This study confirmed the reproducibility of the workflow through repeated trials at different postnatal time points and across the distinctive ILMs. High-throughput diagnostics from 90 RNA samples indicated no sequencing alignment scores below 70%, validating the extraction strategy. Significant differences between the standard and experimental conditions suggest circumvented challenges and broad applicability to other skeletal muscles. This investigation remains ongoing given the prospect of therapeutic insights to voice, swallowing, and airway disorders. The present methodology supports pioneering global transcriptome investigations in the larynx previously unfounded in literature.

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confidence: 63%

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confidence: 94%

mentioning

confidence: 99%

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An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model

Kemfack

Hernández-Morato

Moayedi

et al. 2022

Sci Rep

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“…Obtained values for RNA range from 20 to 280 ng/mg for different methods in this study are similar to those from skin, heart, or prostate and significantly lower than tissues like muscle, liver, or pancreas. The average DNA yield ranges from 70 to 428 ng/mg for methods compared in this study, which makes it several times less than the yield obtained from muscle, liver, lung, or thymus ( Walker et al, 2016 ; Liska & Ruprecht, 1999 ; Biase et al, 2002 ; Ginzberg, Kafri & Kirschner, 2015 ; Stroh et al, 2021 ). Broad ranges of nucleic acid yield are mainly a result of the capabilities of the techniques used.…”

Section: Discussionmentioning

confidence: 86%

“…The least degraded DNA samples were also achieved with spin-column methods, although additional homogenization in liquid nitrogen (method IV) may cause higher degradation. Method III gives a higher DNA yield (340.70 (284.60–394.70) ng/mg) than the spin-column method tested by Stroh et al (2021) , which used homogenization with a disposable plastic pestle and DNeasy Blood & Tissue kit (52 ± 14 ng/mg.). The A260/280 ratio for method III is 1.83 (1.80–1.83), which is exactly in the desired range when A260/280 for the technique used by Stroh et al (2021) is slightly above (2.4 ± 0.2).…”

Section: Discussionmentioning

confidence: 98%

A comparison of five methods to maximize RNA and DNA isolation yield from adipose tissue

Dabrowski,

Rasmus,

Jundzill

et al. 2024

PeerJ

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Adipose tissue in the human body occurs in various forms with different functions. It is an energy store, a complex endocrine organ, and a source of cells used in medicine. Many molecular analyses require the isolation of nucleic acids, which can cause some difficulties connected with the large amount of lipids in adipocytes. Ribonucleic acid isolation is particularly challenging due to its low stability and easy degradation by ribonucleases. The study aimed to compare and evaluate five RNA and DNA isolation methods from adipose tissue. The tested material was subcutaneous porcine adipose tissue subjected to different homogenization methods and RNA or DNA purification. A mortar and liquid nitrogen or ceramic beads were used for homogenization. The organic extraction (TriPure Reagent), spin columns with silica-membrane (RNeasy Mini Kit or High Pure PCR Template Preparation Kit), and the automatic MagNA Pure system were used for the purification. Five combinations were compared for RNA and DNA isolation. Obtained samples were evaluated for quantity and quality. The methods were compared in terms of yield (according to tissue mass), purity (A260/280 and A260/230), and nucleic acid degradation (RNA Integrity Number, RIN; DNA Integrity Number, DIN). The results were analyzed statistically. The average RNA yield was highest in method I, which used homogenization with ceramic beads and organic extraction. Low RNA concentration didn’t allow us to measure degradation for all samples in method III (homogenization with ceramic beads and spin-column purification). The highest RNA quality was achieved with method IV using homogenization in liquid nitrogen and spin column purification, which makes it the most effective for RNA isolation from adipose tissue. Required values of DNA yield, purity, and integrity were achieved only with spin column-based methods (III and IV). The most effective method for DNA isolation from adipose tissue is method III, using spin-columns without additional homogenization.

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Low adipose tissue index as an indicator of hepatic encephalopathy in cirrhotic patients following transjugular intrahepatic portosystemic shunt

et al. 2023

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Human adipose and skeletal muscle tissue DNA, RNA, and protein content

Cited by 12 publications

References 63 publications

An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model

An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model

A comparison of five methods to maximize RNA and DNA isolation yield from adipose tissue

Low adipose tissue index as an indicator of hepatic encephalopathy in cirrhotic patients following transjugular intrahepatic portosystemic shunt

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