I would like help reading the quality and integrity of my RNA. This picture makes me think all my RNA seems degraded?
I have been trying to extract RNA from mouse lung tumor and normal tissue for the past month with varied success in terms of concentrations and purity from Nanodrop and Qubit (concentrations too low to measure to 80 ng/uL). My tissue sizes are very small (about 7 to 20mm3), so I've been trying to do all I can to maximize my yield like using RNALater-ICE and using RNAse Zap on my equipment. I currently use an Omni tissue homogenizer for 40s on my tissue in RLT buffer plus beta-mercaptoethanol. My extractions have been done with the Qiagen Mini kit and I've read all the posts I can find on here and several papers about how to optimize RNA yield with this kit; yet my yields are just averaging about 40 ng/uL of RNA.
I've decided to add the optional DNAse digestion step to my extractions and I wanted to check for gDNA contamination and assess the RNA quality of my extractions by gel electrophoresis since we do not have access to a Bioanalyzer. I've seen that RNA can be run on native gels, so these pictures are of a 1% agarose gel with 1X TBE and 60V at 60 minutes with a DNA ladder to check running of the gel and my RNA samples +/- DNase, samples were mixed with 6x loading dye. Are these images indicative of RNA degradation or do I need to run a non-denaturing gel (if so, how do I do that)? There's a dark pinkish band on the bottom half of the gel that's hard to see in the picture very clear in normal lighting and I'm not sure what that represents? I definitely don't see bands for 28S and 16S so I'm feeling kind of hopeless that all my RNA quantities measured by Qubit represent poor quality RNA. I would like to send my RNA for RNA sequencing eventually (not specifically from these samples, which are more for practice).
Thanks so much in advance for reading through and offering any guidance.
I'm a bit confused. You say the gel is a 1% agarose gel with 1X TBE which describes a non-denaturing gel but later you inquire about running a non-denaturing gel? Did you add a denaturant to the gel you show pictures of?
The RNA looks degraded since there is possibly only 1 ribosomal RNA band and there should be 2.
The purple (dark pink) front in the gel is the bromophenol blue dye which is part of the loading dye.
Tom Masi Thanks for the response- sorry I confused non-denaturing with denaturing, I meant to ask if I should try a denaturing gel or at least add formaldehyde/heat up my samples to see if that would improve anything?
Or is this evidence enough that all my samples are degraded? Quite lost at the moment because I'm not sure what I can else optimize
Thanks for the clarification.
Don't waste your time with a denaturing gel. The results will still be the same. Degraded RNA.
If you just want to check the integrity of the RNA use a non-denaturing gel. But heat/boil/100 degrees C for 5 minutes to denature the RNA, then add loading dye and load the gel. If you can, load the gel with the current running through it.
Lung tissue is a very poor source of RNA to begin with compared to something like liver. Can you homogenize multiple lungs/tissue samples together such that you increase the overall yield?
Tom Masi Thanks for the suggestion. I will try heating some of my others samples and running it on a non-denaturing gel.
Do you mean to run different pieces of the same tumour through different RNA extraction columns and then combine the RNA? The Qiagen kits ask for not more than 30 mg of tissue on their column and I'm basically maxing that out. These are also old samples collected a couple years ago stored in our -80 so maybe that has contributed to the sample degradation?
Yes you could combine the RNA from different columns together.
The Qiagen kit also says you should get 10-20ug of RNA from lungs, assuming 30mg. However, the binding capacity for the column is 100ug. So in theory, you could use 150-300mg tissue and still be under the max binding capacity.
Depending on how the samples were frozen years ago, flash freeze in liquid nitrogen then store at -80 vs just putting them in the -80 to freeze, could definitely contribute to degradation.
Thanks so much- this is very helpful. I highly appreciate your help- I will give that a try! - Peipei
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