TITLE: High-Yield Skeletal Muscle Protein Recovery from TRIzol® after RNA and DNA Extraction
AUTHORS: Yuan Wen, Ivan Vechetti , Taylor Valentino, John J McCarthy

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Extraction of DNA, RNA, and protein from the same sample would allow for direct comparison of genomic, transcriptomic, and proteomic information. Commercially available kits exhibit poor protein yield and TRIzol® reagent produces a protein pellet that is extremely difficult to solubilize. In response to these limitations, this study presents an optimized method for the extraction of protein from the organic phase of TRIzol® reagent that allows for higher yield recovery of skeletal muscle protein compared to direct homogenization in a common protein lysis buffer. The presented method is inexpensive, simple and fast; requires no additional treatment of the protein pellet for dissolution; and is compatible with downstream western blot applications.</div></div>

[STEPS]
?. [RNA Extraction]
Mince muscle tissue into small (~10 mg) pieces.
?. [RNA Extraction]
Isolate RNA using TRIzol® reagent, following manufacturer’s instructions.Important point: Use 0.5 ml of TRIzol® reagent per piece (Important point: For better phase separation, use 100 µl of BCP instead of Chloroform.
?. [RNA Extraction]
Remove top RNA fraction (aqueous phase) according to manufacturer’s recommendation.
?. [RNA Extraction]
Follow manufacturer’s instructions for RNA precipitation, wash, and re-suspension or choice of RNA column purification kit.
?. [RNA Extraction]
Proceed to DNA precipitation.
?. [DNA Precipitation]
Precipitate DNA following TRIzol® manufacturer’s instructions.
?. [DNA Precipitation]
Wash and re-suspend DNA pellet according to manufacturer’s instructions or choice of genomic DNA column purification kit.
?. [DNA Precipitation]
Proceed to Protein Recovery.
?. [Protein Recovery]
Transfer an aliquot (~0.45 ml) of protein-TRIzol®-EtOH solution into 2 ml tube.
?. [Protein Recovery]
Add 650 μl of 100% EtOH (protein precipitation step) to protein-containing phenol–ethanol supernatant.
?. [Protein Recovery]
After vortexing the samples, mix with 100 μl of BCP and vortex again.
?. [Protein Recovery]
For phase separation, add 600 μl water and vortex vigorously.
?. [Protein Recovery]
Centrifuge at 12,000g for 5 minutes at room temperature.
?. [Protein Recovery]
Remove and discard the upper aqueous layer (proteins are in the interphase).
?. [Protein Recovery]
Add 700 μl of 100% EtOH to the remaining interphase and BCP phase.
?. [Protein Recovery]
Vortex samples vigorously and centrifuge at 12,000g for 5 minutes to pellet the protein.
?. [Protein Recovery]
Remove supernatant and dry the pellet at room temperature for 1-2 minutes.
?. [Protein Recovery]
Add desired volume of SDS-Urea Buffer (at least 100 μl per 10 mg tissue), vortex, and allow protein pellet to dissolve at room temperature.
?. [Protein Recovery]
To quantify protein concentrations, follow the manufacturer’s protocol for the Bio-Rad RC/DC Protein Assay (5000121).Important point:The RC component of the protein assay is necessary due to the presence of reducing agents in the SDS-urea buffer.Critical Step:Protein samples in the SDS-urea buffer are diluted 10-fold to reduce the total starting amounts of protein and reducing agents in the assay. Protein standards are made using bovine serum albumin solubilized in the respective buffer and are subjected to the same dilution step as the unknown samples ensure consistency.