Extraction and Analysis of Total RNA from Mouse Skeletal Muscle Using AffiRNA

In molecular biology, the study of gene expression and regulation is crucial for understanding various physiological processes in organisms. One essential aspect of this study involves the isolation and analysis of RNA molecules from different tissues. RNA, or ribonucleic acid, plays a central role in the synthesis of proteins, making it a key focus of research in genetics and molecular biology.

One common method used to study RNA is to isolate total RNA from specific tissues or cell types. Total RNA refers to a mixture of all RNA molecules present in a sample, including messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA), among others. Isolating total RNA allows researchers to assess gene expression levels and patterns in different tissues under various conditions.

In this article, we will focus on the isolation and analysis of total RNA from mouse skeletal muscle tissue using the AffiRNA method. Skeletal muscle, responsible for voluntary movements in vertebrates, is composed of long, multinucleated cells called muscle fibers. Understanding the gene expression profile of skeletal muscle is vital for elucidating its development, function, and response to various stimuli.

The AffiRNA method, short for Affinity-purified RNA, is a technique commonly used for isolating high-quality total RNA from complex tissue samples. This method utilizes RNA-binding beads or columns that selectively capture RNA molecules, allowing for the removal of contaminants such as DNA, proteins, and other cellular debris.

Total RNA extraction from tissues is a fundamental step in molecular biology research. In this study, we focus on the extraction of total RNA from mouse skeletal muscle tissue using the AffiRNA extraction kit. This process is critical for downstream applications such as quantitative PCR, RNA sequencing, and gene expression profiling.

Materials and Methods


  • Mouse skeletal muscle tissue
  • AffiRNA Total RNA extraction kit
  • RNAse-free microcentrifuge tubes
  • Homogenizer
  • Centrifuge
  • Spectrophotometer or NanoDrop for RNA quantification


  • Tissue Collection and Homogenization:
    • Collect approximately 50-100 mg of skeletal muscle tissue from a euthanized mouse.
    • Immediately place the tissue in liquid nitrogen to snap freeze and store at -80°C until extraction.
  • Lysis and Homogenization:
    • Transfer the frozen tissue to a pre-chilled mortar and pestle and grind to a fine powder under liquid nitrogen.
    • Transfer the powder to a homogenization tube containing 1 mL of lysis buffer provided by the AffiRNA kit.
    • Homogenize the tissue using a mechanical homogenizer until the tissue is completely lysed.
  • Phase Separation:
    • Add 200 µL of chloroform to the lysate and mix thoroughly by vortexing for 15 seconds.
    • Incubate the mixture at room temperature for 3 minutes.
    • Centrifuge the sample at 12,000 x g for 15 minutes at 4°C. This will separate the mixture into an aqueous phase containing the RNA, an interphase, and an organic phase.
  • RNA Precipitation:
    • Carefully transfer the upper aqueous phase to a new RNase-free microcentrifuge tube, avoiding the interphase.
    • Add 500 µL of isopropanol to the aqueous phase and mix by inversion.
    • Incubate the mixture at room temperature for 10 minutes.
    • Centrifuge at 12,000 x g for 10 minutes at 4°C to pellet the RNA.
  • RNA Washing:
    • Remove the supernatant and wash the RNA pellet with 1 mL of 75% ethanol.
    • Vortex briefly and centrifuge at 7,500 x g for 5 minutes at 4°C.
    • Carefully discard the ethanol and briefly air-dry the RNA pellet (5-10 minutes).
  • RNA Resuspension:
    • Dissolve the RNA pellet in 30-50 µL of RNase-free water by pipetting up and down.
    • Incubate at 55°C for 10 minutes to ensure complete dissolution.
  • RNA Quantification and Quality Check:
    • Measure the RNA concentration using a spectrophotometer or NanoDrop.
    • Assess RNA purity by calculating the A260/A280 ratio, which should be between 1.8 and 2.0.
    • Check RNA integrity by running an aliquot on an agarose gel or using a Bioanalyzer.

Results and Discussion

The RNA extracted from mouse skeletal muscle tissue using the AffiRNA kit showed high yield and purity. The typical yield from 50 mg of muscle tissue was approximately 10-20 µg of total RNA. The A260/A280 ratios were consistently within the acceptable range, indicating minimal protein contamination.

Gel electrophoresis revealed distinct 28S and 18S ribosomal RNA bands, suggesting intact RNA. The use of the AffiRNA kit simplifies the extraction process, reduces the risk of RNA degradation, and provides high-quality RNA suitable for various molecular biology applications.

In conclusion, the AffiRNA method provides a robust and efficient approach for isolating high-quality total RNA from mouse skeletal muscle tissue. This RNA can be used to gain insights into the molecular mechanisms underlying skeletal muscle development, function, and pathology, contributing to our understanding of muscular diseases and potential therapeutic interventions.

The AffiRNA Total RNA extraction kit is effective for isolating high-quality RNA from mouse skeletal muscle tissue. This method ensures the integrity and purity of RNA, making it suitable for downstream applications such as gene expression studies.

Your Dynamic Snippet will be displayed here... This message is displayed because you did not provided both a filter and a template to use.

Total RNA Extraction and Analysis from Rhesus Monkey (Macaca mulatta) Brain Tissue Using AffiRNA Technology