I extracted RNA from single cells using RNAeasy kit. I did add DNase I as stated in the extraction protocol. However my negative control (without reverse transcriptase) still showed amplification from the qPCR suggesting all the DNA was not digested. Is okay to simply add DNase I to each of my RNA samples for further digest of the DNA and then heat kill the DNase I?
1. Will this likely have an adverse effect on my RNA quality?
2. Will it affect my qPCR process?
Yes, DNase can be added to extracted RNA solution, incubated at RT and enzyme heat killed. This is a common method for removing DNA contamination from RNA samples. The DNase will digest any DNA that is present in the sample, and the enzyme can then be heat killed to inactivate it. This method is relatively simple and inexpensive, and it is effective for removing low levels of DNA contamination. However, it may not be effective for removing high levels of DNA contamination. In these cases, it may be necessary to use a more rigorous method for removing DNA contamination, such as column-based purification or on-column DNase treatment.
Yes, it is okay to simply add DNase I to each of your RNA samples for further digest of the DNA and then heat kill the DNase I.
DNase I is an enzyme that can degrade RNA, so it is important to use the correct concentration of DNase and to incubate the sample for the correct amount of time. It is also important to use a DNase that is free of RNases.
Yes, DNase I can affect your qPCR process.
To prevent DNase I from degrading the DNA template, it is important to heat kill the DNase I after it has been used to digest the DNA contamination. This can be done by incubating the sample at 65°C for 15 minutes.
Yes, DNase can be added to an extracted RNA solution, incubated at room temperature, and then heat killed. However, it is important to note that heat killing of enzymes usually requires higher temperatures than room temperature. Therefore, it is recommended that you heat the solution to a temperature between 70-80°C for at least 10 minutes in order to ensure complete inactivation of the enzyme.
It's standard practice to DNase treat you RNA before starting your qPCR experiment.
Did you run out the reactions on a gel to check the product size when you got amplification in your noRT negative control? Assuming your primers amplify different size products from gDNA vs cDNA, then you can see if it's gDNA or if you have an issue with contamination.
Good luck!
No, DNase enzyme should not be added directly to the extracted RNA solution. Addition of DNase enzyme typically requires a specific set of conditions for activity, such as specific pH and salt concentrations, as well as a certain incubation temperature. After addition of DNase, the sample should be incubated at the specified temperature and then heat inactivated to stop enzyme activity.
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