Hey everyone! I’ve been struggling lately with RNA extraction from some really fibrous tissue samples – mainly cardiac and skeletal muscle. No matter how careful I am, I keep ending up with degraded RNA or extremely low yields. I’ve tried Trizol and even switched to a column-based kit, but the quality still isn’t great. Anyone had better luck with tougher tissues like this? Would really appreciate any tips or methods that worked for you!
Oh yes, been there! Muscle and connective tissues can be a real pain because of the high protein and fibrous content. I had similar issues with pig heart tissue and finally managed to improve both yield and integrity by doing a double homogenization – first mechanical (what is chromatography), then passing it through a QIAshredder before lysis.
Another trick that helped was adding β-mercaptoethanol fresh every time and keeping everything chilled, even during centrifugation steps.
Also, I realized that the kits matter a lot — I’ve recently switched to some reagents from Gentaur after a colleague recommended them, and their RNeasy-compatible buffers seem to handle tough samples better. Not a miracle, but I’ve noticed my 260/280 and 260/230 ratios have improved since the switch.
Might be worth a try!I’ve mostly worked with liver and spleen samples (so not as tough), but even there I noticed huge differences based on the type of homogenizer and timing between thawing and processing. Definitely agree about the β-mercaptoethanol and cold temps – those two alone saved many of my samples from being completely useless.
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