Development and validation of most efficient RNA isolation method from buffalo bull spermatozoa

Area of Science:

• Reproductive Biology
• Molecular Biology
• Bioinformatics

Background:

• Sperm cells present significant challenges for RNA isolation due to low concentration and high fragmentation.
• Previous attempts to isolate RNA from buffalo sperm have explored various methods with limited success.

Purpose of the Study:

• To compare the efficacy of different RNA isolation methods for buffalo bull sperm.
• To identify the optimal protocol for obtaining high-quality and high-concentration sperm RNA.

Main Methods:

• Evaluation of both non-membrane and membrane-based RNA isolation techniques.
• Comparison of TRIzol, heat-lysed TRIzol (H-TRIzol), and a cocktail lysis buffer (C-TRIzol) method.
• Assessment of T-RLT and RLT-T lysis reagent combinations for RNA yield and quality.

Main Results:

• The heat-lysed TRIzol (H-TRIzol) method demonstrated superior performance among conventional techniques.
• The combined T-RLT lysis protocol yielded the best RNA quality and quantity among membrane-based methods.
• T-RLT outperformed RLT-T, showing reduced genomic DNA contamination and membrane clogging.

Conclusions:

• The H-TRIzol method is recommended for its ease of use and superior RNA yield and quality from buffalo sperm.
• This comparative study provides valuable insights for selecting the best sperm RNA isolation protocol for transcriptome and other downstream applications.
• Optimized sperm RNA isolation is crucial for advancing studies in buffalo reproductive biology and genetics.