I am currently facing a challenge related to the extraction of total RNA from 40 mg colon tissue, and I would greatly appreciate your expertise and insights in troubleshooting the issue.
During the total RNA extraction process, I obtained an A260/A280 ratio of 2.48 with 1500 ng/ul concentration as measured by the Nanodrop spectrophotometer. Although this ratio suggests minimal protein contamination, it is higher than the typical range for RNA, raising concerns about the accuracy of the measurement. Furthermore, when I analyzed the extracted RNA samples using agarose 1% gel electrophoresis, I observed a smear pattern, and the 28s and 18s bands appeared very weak in intensity. This indicates potential issues with the integrity and quality of the RNA samples. I am reaching out to the research community for guidance on the possible causes of these observations. Why did I obtain a high A260/A280 ratio, and could this be indicative of an underlying issue with the RNA sample or the extraction process? Additionally, what factors may contribute to the weak 28s and 18s bands and smear pattern, and how can I improve the quality and integrity of the RNA samples to ensure reliable results?
I should add some more details:
I extracted the RNA with Trizol and run the electrophoresis for 40 min with 100 volts.
If your purification kit uses Guanidine thiocyanate as a chaotrophic agent and this is not well washed off the column then GT absorbs at 260 so it looks like you have a lot of dna but really it is GT. Try an extra was with wash buffer in the kit. It will also increase the 260/280 ratio as the od260 is artificially high
Protein contamination causes a high A260/A280 ratio value, and you also observed the effect on the gel due to protein contamination.
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