Some tissues are tricky to work with. This truth was lost on me in the early years of grad school because I worked with liver samples. If you’re extracting RNA from liver samples, you’re likely not losing sleep over your massive RNA yields. But for the folks doing RNA extractions with less willing donors, such as adipose and pancreatic tissue, you may have found yourself lifting up your eppendorf tube to the skies in Oliver Twist-esque pleas for more.
Over time I’ve learned some more practical ways to get better extractions.
Here are few tried and tested tips to increase RNA yield.
1. Follow the protocol (applies to all tissues)
This no-brainer-sounding tip is the most obvious, yet most likely to get you in trouble. If your protocol calls for keeping the tissue on dry ice until homogenization and you’ve just run out, you’re better off going for a long lunch break than opting for regular ice.
With tricky tissues, brief, barely visible thawing contributes to the degradation of your precious RNA. Similarly, if it says “use 75% ethanol…” during a binding or wash step, make sure the vial doesn’t say “50% ethanol”.
2. Get rid of RNAse (applies to nuclease-rich tissues like pancreas and spleen)
Nothing is worse than when the tissue you’re trying to extract RNA from is chewing up all the RNA you’ve been working so hard to get.
Being a digestive tissue, the pancreas contains a large amount of RNAse, the enzyme that degrades RNA. RNA yields can be abysmally tiny if care is not taken to get rid of this endogenous RNAse. One way to do this is to powerfully lyse the cells with a guanidinium-containing solution after tissue collection that will denature the nucleases. If you’re like me, you like to keep tissues waiting in the -80°C freezer for weeks or months before you attend to them, and in that case, snap-freezing (immediate freezing) in liquid nitrogen upon tissue collection helps keep RNAses inactive until they can be lysed.
3. Modify your sample size (applies to adipose tissues)
While 50 mg of liver or skeletal muscle might give you an impressive yield of RNA, the same amount of adipose tissue could leave you playing a not so thrilling game of hide-and-seek. As fat tissue contains a ton of fat (some things in science are just that easy to understand), the amount of other macromolecules in the tissue, such as protein and nucleic acid, is diminished. One way to improve your yield is to use a bigger chunk of tissue.
4. Get rid of the fat cake (applies to fatty tissues like adipose and brain)
If you’re working with adipose tissue, your protocol should include a centrifuge step to separate off a layer of oil (Hence, fat cake. I’m serious—this is how it was referenced in our lab’s protocol).
This step should take place after homogenization in a lysis buffer, and prior to binding the RNA to a column. Brain is another fatty tissue that might need that extra oil-removal step.
Freeze-thaw—you know it’s bad for your samples, don’t you? While working in the lab, you have most likely heard someone say ‘aliquot your protein/cells/DNA/RNA to avoid too many freeze-thaw cycles.’ But do you actually understand why? You probably thought that avoiding freeze-thaw cycles had something to do with damaging cell structure as well as proteins […]
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