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How The Human Mind Distinguishes Between Limbs- RNA Seq Has The Answer!

 The health of the human skeletal muscle is vital for good and quality life and it also predicts several diseases like cancer, diabetes and several other chronic diseases. Skeletal muscles are the tissue that is present in human limbs and is responsible for studying mechanisms behind various diseases and the adaptive effects of many controlled interventions. For mechanistic studies like these, the profile of gene expression in many different states is imperative. The transcriptome of baseline has not been examined systematically, therefore, the purpose behind these studies is the provision of a deep reference profile of male and female skeletal muscles. In the following study, the data from RNA sequencing was to analyze from a large set of 45 human biopsies. The researchers provide extensive information on the transcriptome, which include the 5 previously protein-encoding unannotated transcripts.


Since the global transcriptional issue has been sky-rocketing within the contralateral leg and specific muscles.  The scientist has identified more than 23,000 known isoforms and discovered more than 5000 isoforms, which are different in both the sexes. The transcriptome of both female and male was found rich in genes that are associated with the protein-catabolic process and oxidative metabolism, respectively. The data from the study conducted by scientists demonstrate a very high homogeneity in tissue and it provides an extensive and deep baseline reference for the skeletal muscles transcriptome of human skeletal muscles. This is all regarding novel transcripts, the difference in sexes in the functional ontology.  


The gene expression analysis 


The gene expression has turned into a central element in the study of mechanisms involved in cell regulations and tissue. In terms of study, human skeletal muscles are relatively unique, because they are also accessible for sampling in some meaningful quantities that are easily subjected to control intervention and physiological relevance.  This constitutes the human body’s largest tissue and is found to have a very active metabolism. More than over a past decade, there have been many studies on skeletal muscles, so that the transcriptional effects could be determined on various kinds of unloading and exercise training. It is also a common occurrence in diseases like muscular dystrophies, diabetes and atrophy.  However, the global baseline transcriptome of human skeletal muscles has not been analyzed systematically. 


There is alternative splicing, which significantly extends the transcriptomic, and therefore, the proteomic complications. This has been a very intense area of research and nearly 95% of human genes depict various isoforms and the specific alternative splicing events are the major key-contributors to phenotype specific to tissue. Another important factor that influences the isoform expression is the sex of the subject being analysed, this is also in direct relation to alternative splicing that has only been investigated for either human liver tissue or mouse live tissue. 


The baseline sex differences have also been depicted in muscles of biceps, but the recent investigation is the first one to study the global transcriptome differences have also been depicted by Liu. However, the current findings have revealed the global transcriptome differences between the skeletal muscles of females and males, by using the parallel sequencing technique. 


In a lot of individuals, the samples for study have been acquired from various sites within belly muscles and also in the contralateral muscle. The bottom line is that the sampling sites had the same biochemical, physiological and histological transcriptional properties. All the differences in gene expression stem out of the intervention itself and not because of the heterogeneity found in the tissues. Many studies have declared this assumption is problematic, as in this study only a single cell muscle is shown to be heterogeneous with different depths. This is especially the case regarding the fibre area and fibre type distribution. Furthermore, it also explains the differences in histology and biochemistry of the subjected fibre. 


The difference also might be true for skeletal muscle transcriptome, something that has not yet been investigated in humans at a global level. Moreover, the differences between 2 separate legs of an individual and also the magnitude of interindividual variability are a few basic questions that need to be asked systematically. 



RNA isolation, sequencing and library construction


The total RNA was extracted by the TRIzol method, which is found following the specifications of the manufacturer. The quality and concentration of the RNA were determined with RNA 6000 Nano Chip, found on the 2100 Bioanalyzer automated electrophoresis system. For experimental purposes, the researchers took two micrograms of total RNA for the library preparation of every sample. These samples were then subsequently, prepared and bar-coded by using automated platforms. 

The libraries were crowded on a cluster-generation system with the cluster-generation kit and Illumina HiSeq paired-end. In the experiment, every lane was spiked with 1 % phiX control library. 


The general expression profile of skeletal muscles in humans


The transcriptome analysis of skeletal muscles was done via RNA-sequence on muscle biopsies from 18 numbers of individuals, with 9 females and 9 male. The sequencing reads of paired-end were all aligned to human genomes. The normalized expression of the gene gets calculated as FPKM. As the expectations go, the majority of the samples are clustered by an individual. Scientists found 12,659 numbers of transcripts that get expressed in human skeletal muscles, by using an average cutoff method for FPKM which is greater than 1. 

The sequence data of RNA also has correlations to microarray expression data from an estimate of 25 skeletal muscle samples from a different published study. The correlation of Spearman between the levels of expressions was imperative at both transcript levels and gene. 


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Gene expressed in human skeletal muscles 


The analysis of the expression in skeletal muscle identifies nearly 23,002 known isoforms. The average value of isoform in every gene is 2.12. However, 113 genes had expressed over 10 ENSEMBL annotated isoforms in the baseline of skeletal tissues according to the findings of Cufflinks analysis. To closely investigate more isoforms, the abundant isoforms were found high through every subject, while more low-expression isoforms are found to be more variable. Besides, no isoform showed expressions patterns that are specific to an individual (Lindholm, n.d).



Lindholm, M. E., Huss, M., Solnestam, B. W., Kjellqvist, S., Lundeberg, J., & Department of Physiology and Pharmacology, Karolinska Institute, Solna Sweden. (n.d.). The human skeletal muscle transcriptome: sex differences, alternative splicing, and tissue homogeneity assessed with RNA sequencing. The FASEB Journal, 1(1).

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