High-yield Skeletal Muscle Protein Recovery from TRIzol After RNA and DNA Extraction

Wen, Yan; Vechetti, Ivan J.; Valentino, Taylor; McCarthy, John J.

doi:10.2144/btn-2020-0083

Cited by 15 publications

(11 citation statements)

References 29 publications

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“…Protein was extracted from the organic phase of TRI Reagent following RNA extraction using the optimized protocol (Wen et al 2020). Briefly, following the final step of ethanol addition, samples were centrifuged and the resulting protein pellet solubilized using SDS-urea buffer (100 mm Tris, pH 6. with sample processing resulted in n < 10 for qRT-PCR data in some instances (see Results).…”

Section: Western Blottingmentioning

confidence: 99%

Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise

Figueiredo

Wen

Alkner

et al. 2021

The Journal of Physiology

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Ribosome biogenesis and MYC transcription are associated with acute resistance exercise (RE) and are distinct from endurance exercise in human skeletal muscle throughout a 24 h time course of recovery.r A PCR-based method for relative ribosomal DNA (rDNA) copy number estimation was validated by whole genome sequencing and revealed that rDNA dosage is positively correlated with ribosome biogenesis in response to RE.r Acute RE modifies rDNA methylation patterns in enhancer, intergenic spacer and non-canonical MYC-associated regions, but not the promoter.r Myonuclear-specific rDNA methylation patterns with acute mechanical overload in mice corroborate and expand on rDNA findings with RE in humans. r A genetic predisposition for hypertrophic responsiveness may exist based on rDNA gene dosage.Vandré C. Figueiredo completed his PhD in Biomedical Sciences at the Liggins Institute, The University of Auckland, under the supervision of Dr David Cameron-Smith before joining the laboratory of Dr John McCarthy at the University of Kentucky for his postdoctoral training in 2016. His research is focused on the role of ribosome biogenesis and the cellular signalling regulating skeletal muscle size. He seeks to understand the mechanism of muscle size determination hoping to apply this knowledge to ameliorate muscle atrophy conditions, with particular interest in ageing, cachexia and muscle disuse.

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Section: Western Blottingmentioning

confidence: 99%

Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise

Figueiredo

Wen

Alkner

et al. 2021

The Journal of Physiology

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“…A non-complete solubilization could alter the correct protein estimation and may represent a limitation for downstream analysis. Indeed, several studies described alternative methods for protein solubilization in order to improve the yield [ 12 , 19 , 21 ].…”

Section: Discussionmentioning

confidence: 99%

“…Finally, the possibility of using the same surgical sample to extract, at the same time, RNA and proteins would be desirable as it would allow a direct comparison of transcriptomic and proteomic information [ 12 ]. Commercially available reagents allow the implementation of these procedures and, since protein extraction is not a trivial undertaking, the possibility of using the whole biological material, without dividing it for different analyses, increases the RNA yield and, thus, contributes to improving the most difficult phase of the procedure.…”

Section: Introductionmentioning

confidence: 99%

A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone

et al. 2022

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Molecular differences between cortical and trabecular bone, of relevance to understanding the pathophysiological basis of bone diseases, can be determined only through effective isolation methods for RNA and proteins. Here we present a TRIzol-based method, which combines bone pulverization and homogenization to extract simultaneously total RNA and proteins from human cortical and trabecular bone from the same carrot. RNA integrity and purity were determined as the 260/280 nm and 260/230 nm absorbance ratios and the 28S/18S rRNA ratio. Protein integrity and quality were evaluated by Coomassie blue staining. Reverse transcription quantitative polymerase chain reaction and immunoblotting for bone-specific genes and proteins were performed to verify the suitability of the isolated material in downstream applications. The 260/280 nm and 260/230 nm absorbance ratios were, on average, less than or equal to 1.8. Bands on agarose gel were consistent with intact RNA, with mean 28S/18S ratios of 1.68 ± 0.35 and 1.88 ± 0.10 for cortical and trabecular bone, respectively. Band patterns after Coomassie blue staining confirmed protein integrity. Successful gene and protein expression analysis, with relevant differences between the two compartments, highlighted the suitability of the material in downstream applications. The method presented here is appropriate and effective for the study of human bone.

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“…All the analyses were performed in triplicate. The cells were lysed, and total RNA was extracted using TRIzol reagent (Takara, Bao Bioengineering (Dalian) Co., Ltd., Dalian, China) according to the manufacturer’s protocol and transcribed into cDNA using a Reverse Transcription Kit (Takara, Bao Bioengineering (Dalian) Co., Ltd., Dalian, China) [ 31 ]. The expression patterns of the target genes and the transcriptional responses of the target genes to the muscle were investigated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR).…”

Section: Methodsmentioning

confidence: 99%

“…RT-qPCR was performed using the ChamQTM Universal SYBR ® qPCR Master Mix (Nanjing novozan Biotechnology Co., Ltd., Nanjing, China) with the following thermal cycling conditions: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. Each experiment was performed independently three times. The relative expression levels of the target genes were normalized to that of β-actin quantification using the 2 − △△Ct method [ 31 ].…”

Section: Methodsmentioning

confidence: 99%

MiR-24-3p Conservatively Regulates Muscle Cell Proliferation and Apoptosis by Targeting Common Gene CAMK2B in Rat and Cattle

Liu

et al. 2022

Animals

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Skeletal muscle plays an important role in the growth and development of meat animals. MicroRNAs (miRNAs) can participate in the regulation of muscle development-related functions; however, there have been few reports on whether there are related miRNAs that conservatively regulate muscle development among different species. In this study, the miRNA transcriptome sequencing data of the muscle tissue of cattle, rat, goat, and pig showed that miR-24-3p may conservatively regulate muscle development in these species. Furthermore, mmu-miR-24-3p can positively regulate C2C12 cell proliferation and apoptosis by regulating key proliferation and apoptosis genes in muscle development, which was verified by CCK-8 and RT-qPCR. Bta-miR-24-3p can also positively regulate the proliferation and apoptosis of bovine muscle primary cells by regulating key proliferation and apoptosis genes in the process of muscle development, as verified by CCK-8 and RT-qPCR. The target genes of miR-24-3p in cattle, rat, goat, and pig, which include a large proportion of target genes shared among the four species, are enriched in multiple cell functions and signal pathways that are closely related to muscle development, as revealed by GO and KEGG enrichment analysis. A double luciferase test showed that the shared target genes WNT4, CAMK2B, and TCF7 were targeted by mmu-miR-24-3p in rat and bta-miR-24-3p in cattle. These three shared target genes WNT4, CAMK2B, and TCF7 are involved in the Wnt signaling pathway, which showed that miR-24-3p plays an important role in rat and cattle. The shared target gene (CAMK2B) in rat and cattle increased significantly after the inhibition of miR-24-3p by RT-qPCR. The findings of this study contribute to a better understanding of the role of miR-24-3p in the regulation of muscle development.

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High-yield Skeletal Muscle Protein Recovery from TRIzol After RNA and DNA Extraction

Cited by 15 publications

References 29 publications

Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise

Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise

A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone

MiR-24-3p Conservatively Regulates Muscle Cell Proliferation and Apoptosis by Targeting Common Gene CAMK2B in Rat and Cattle

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