分子生物学

Skeletal muscle is composed of different cells and myofiber types, with distinct metabolic and structural features. Generally, transcriptomic analysis of skeletal muscle is performed using whole muscle, resulting in average information as all cells composing the organ contribute to the expression value detected for each gene with the loss of information about the distinctive features of each specific myofiber type. Since myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism, it would be beneficial to have fiber-specific information about gene expression. Here, we describe a protocol for the isolation and the transcriptomic analysis of single individual myofibers. The protocol was set up using single myofibers isolated from soleus and Extensor Digitorum Longus (EDL) muscles, but it can be applied to all skeletal muscles. Briefly, muscles are enzymatically dissociated and individually collected. Long RNAs (> 200 nt) and short RNAs (< 200 nt) are separately purified from each myofiber and used to produce libraries for microarray or sequencing analysis. Through this approach, myofiber-specific transcriptional profiles can be produced, free from transcripts from other non-contractile cell types, in order to identify mRNA-miRNA-lncRNA regulatory networks specific for each myofiber type.

This protocol describes the generation and functional validation of microRNA (miRNA) sponge or decoy constructs. When expressed from a strong promoter, these transcripts can sequester specific miRNA:RISC complexes, thereby resulting in a derepression of endogenous target mRNA. Hence, cells expressing such sponges display a partial or full miRNA loss-of-function phenotype.

Depending on the sponge sequence, the activity of any miRNA of choice can be inhibited by sponge sequestration, but it should be noted that these constructs do not seem to be specific for one particular miRNA. Rather, all miRNAs of the same family as defined by the seed sequence will be affected, albeit to a different degree.

microRNA (miRNA) directly associates with its target transcripts (mRNA). This protocol describes a method for detection of direct interaction between miRNA and mRNA. The result of interaction helps screening the specific target mRNAs for a miRNA.