During the electrophoresis run on a 1% agarose gel at 100 volts for 25 minutes, I was unable to clearly visualize the expected 28S and 18S bands representing ribosomal RNA. Instead, a smear-like pattern appeared on the gel. The ladder was run correctly, indicating that the gel and electrophoresis setup were functioning properly.
To provide context, I measured the concentration of my total RNA using a nanodrop spectrophotometer, which yielded a reading of 2.07 in A260/A280. The concentration was determined to be 1352 ng/μl, and for the gel analysis, I loaded 3 μl of RNA with 1 μl of loading buffer and only 3 ul Ladder.
Considering these results, I am unsure about the integrity of my total RNA and whether the observed smear is indicative of degradation or other factors affecting the sample quality. I am reaching out to this esteemed community in the hopes that someone with experience in RNA electrophoresis can offer insights into potential causes for the observed smear and absence of distinct 28S and 18S bands.
Dear Amirhossein, I think your RNA samples are degraded. How did you isolate/store the RNA?
The RNA isolation was performed using a Trizol reagent according to standard protocols. The samples used in this study were colon tissue specimens obtained from surgical procedures. Immediate handling was ensured, with each sample transferred to a liquid nitrogen tank within 30 minutes after surgery. Subsequently, the samples were stored at -80 degrees Celsius for subsequent downstream processes, including RNA isolation. What can I do to maintain the RNA integrity and prevent RNA degradation?
What colour is your Trizol reagent. It should be pink but is light sensitive and may not work well if the dye indicator turns yellow
My trizol reagent is yellow. I didn't get what you mean. could you please explain more?
Trizol contains phenol and an acidity detecting dye. At the right pH this dye is pink Storage or light affects phenol. Its OH group activates the ring structure to allow addition of electrophilic ions such as OH- , This forms quinone which then reacts with 2 molecules of phenol to form phenoquinone which changes both the nature of the phenol and its pH leading to poor rna purification. I think that you will do better with a new bottle of Trizol
https://socratic.org/answers/607619 will show you images of the transformation
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