I am planning to do single-nuclei sequencing for brain organoids. However, the organoids experienced 25 minutes in room temperature prior to freezing. Most of the time they were in their culture medium, but had a wash (0-2 minutes) in room temperature PBS prior to snap freezing in liquid nitrogen vapour. My questions:
1. Would the nuclear RNA be still intact?
2. How much would the exposure to room temperature impact gene expression?
3. What is your experience in filtering out (during analysis) early response transcripts expected to be upregulated due to processing of the samples?
- The samples for the RNA analysis are quick freezed not primarily because RNA degradation at room temperature. Most of the living being survive at room temperature. That clearly mean that RNA in its original environment is temperature stable (not for longer duration, however).
- The primary aim of quick freeze of samples is the get the snapshot of cellular activity freezed. Since any change in the cellular/tissue environment would cause the change in gene expression, every second has its importance.
- It would be obvious if the temperature of sample before and after its collection/removal/dissection is same/constant, the change in gene expression would not be caused by the change in temperature.
- What is your experience in filtering out (during analysis) early response transcripts expected to be upregulated due to processing of the samples?
>> I don't have experience in particular brain related samples, but any change in expression would be caused by the complex interaction of different stimuli a cell can perceive and it would reflect in the RNA expression profile. And this can not be generalized and to my knowledge it is absolutely very hard to fix by the analysis methods/procedures in the absence of prior verified studies and strict control samples.
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