Hello, I'm trying to elaborate a denaturing 12% polyacrylamide gel (19:1) with urea 8M in order to visualize the presence of plasma total miRNAs, I'm using TBE buffer, my extraction (Trizol) concentration have been from 500 to 1000ng/ul of RNA in the Nanodrop, I've loaded from 5-10 ul, and I haven't been able to visualize any band or cloud near the 25 nt mark in my ladder, I've also tried to denatured my sample at 95 degrees Celsius and without. I also have tried to run the gel without urea, and with higher concentrations of polyacrylamide. Nothing has worked. Has someone done this protocol successfully that could help me?
The PAGE (Polyacrylamide Gel Electrophoresis) with urea technique is commonly used for assessing the integrity of small RNA molecules, including microRNAs (miRNAs). Here's how you can perform PAGE with urea for miRNA analysis:
Materials:
Procedure:
Interpretation:
- Intact miRNAs will appear as sharp, discrete bands on the gel.
- Degraded or partially degraded miRNAs may exhibit smearing or a ladder-like pattern, indicating fragmentation.
- The absence of bands suggests either RNA degradation or issues with sample preparation.
By performing PAGE with urea, you can assess the integrity of miRNAs in your samples, which is crucial for downstream miRNA expression analysis and other functional studies.
Not for miRNA but I used Urea gels for RNA.
1. What dye do you use to stain your gel? I used SybrGold.
2. Do you have a size marker and can you see it when imaging the gel?
3. Nanodrop concentration can be misleading since it won't differentiate RNA from DNA or nucleotides. Do you have access to flourometric measuring instrument such as Qubit?
1. Silver
2. yes, 25 nt DNA ladder
3. Yes, I do have access to a Qubit, I'm going to use it in two weeks.
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