It is generally recommended to follow the manufacturer's protocol for using RQ1 RNase-Free DNase (Promegt) for RNA extraction samples. This includes the step of incubating the samples at 65°C for 10 minutes to inactivate the DNase.

The purpose of the DNase treatment is to remove any contaminating genomic DNA that may be present in the RNA samples. This is important because genomic DNA can interfere with downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR) or other RNA analysis methods.

The 65°C incubation step is important because it helps to inactivate the DNase and prevent further degradation of the RNA. Skipping this step could result in incomplete inactivation of the DNase, which could lead to further degradation of the RNA and potentially affect the accuracy of your results.

Therefore, it is generally not recommended to skip the 65°C incubation step when using RQ1 RNase-Free DNase (Promegt) for RNA extraction samples. If you have experienced issues with unclear bottom bands on gel electrophoresis after performing the DNase treatment, there may be other factors contributing to this problem. It may be helpful to consult with a scientist or technical support representative for more guidance on troubleshooting and optimizing your RNA extraction and analysis procedures.

Thank you dear Bertrand Cornu for comprehensive answer.

I had to change to use another Kit (DNase 1 of Thermo scientific) because of time limit.

In conclusion, it was the best kit that l experienced for treatment my RNA extraction. The result on gel electrophoresis was great.

The step you are referring to in the protocol, which involves incubating at 65°C for 10 minutes to inactivate the DNase, is crucial for effectively stopping the DNase activity and preventing any residual DNase from interfering with downstream applications. While the elevated temperature may affect the stability of your RNA, it is necessary to ensure complete inactivation of the DNase enzyme.

However, if you have concerns about the stability of your RNA at 65°C, there are alternative methods to inactivate DNase without subjecting your samples to high temperatures. One commonly used approach is to use a DNase Inactivation Reagent that effectively removes and inactivates DNase enzymes without the need for heating. This reagent can be added to the reaction mix after the DNase digestion step, and the mixture can be directly used for downstream applications.

Before omitting the inactivation step or using alternative methods, it is important to thoroughly validate and optimize the modified protocol to ensure reliable and accurate results. It is recommended to perform control experiments to verify the effectiveness of the modified DNase treatment and assess the integrity and quality of your RNA after the modification.

Remember, the inactivation step is included in the protocol for a reason, as it helps prevent residual DNase activity from degrading or contaminating your RNA samples. Therefore, it is generally recommended to follow the protocol as closely as possible to obtain reliable and reproducible results.

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Thank you dear Nikolay Klyashtornyy for your advices.

You have very good experiences in RNA.

yours sincerely