Computational protocol: Alleviating exercise-induced muscular stress using neat and processed bee pollen: oxidative markers, mitochondrial enzymes, and myostatin expression in rats

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Protocol publication

[…] Analytical standards such as gallic acid, quercetin, kaempferol, Folin-Ciocalteu phenol reagent, diammonium sulfate, meta-phosphoric acid, ortho-phosphoric acid, analytical reagent (AR) grade solvents ethanol, ethyl acetate, and methanol were procured from Merck, New Jersey, USA. Captex 355 was a generous gift sample from Abitec Corp. Cleveland, OH, USA. Polyoxyethylene sorbitan monolaurate (Tween 80) AR grade was purchased from Merck. An RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) kit was purchased from Biotools B&M Labs, Madrid, Spain. The primers myostatin and β-actin were synthesized and purchased from Amnion Biosciences Pvt. Ltd., Bengaluru, India. All the reagents and solvents used in this study were AR grade. [...] The RNA extraction and RT-PCR analysis was performed as per a previous study using a standard protocol provided by Biotools B&M Labs. Briefly, the isolated gastrocnemius muscle tissue (30–40 mg) was disrupted in liquid nitrogen using a mortar and pestle. To this, lysis buffer (350 μL) and β-mercaptoethanol (3.5 μL) were added followed by vigorous vortexing. The lysate was loaded into a filtering column in a collection tube and centrifuged for 1 minute at 11,000 g. The clarified lysate was mixed with 70% ethanol (350 μL) and loaded into the RNA binding column in a collection tube followed by centrifugation for 30 seconds at 11,000 g. Desalting buffer DBR (350 μL) and rDNase reaction mixture (95 μL) were added and incubated at room temperature for 15 minutes. Following successive washings with wash buffer, RNA was resuspended in ribonuclease-free water (60 μL) and centrifuged. The elute containing pure RNA was stored at −80 °C until analysis. […]

Pipeline specifications

Software tools Biotools, MUSCLE
Applications Population genetic analysis, Nucleotide sequence alignment
Organisms Apis mellifera, Brassica juncea, Rattus norvegicus
Chemicals Glutathione, Malondialdehyde, Nitric Oxide, Polysorbates, Superoxides, Polyphenols