Sampling for transcriptome analysis
Hi all,
I am going to do a transcriptome analysis of small scale S. cerevisiae fermentations using RNA-seq. I would like to get some feed-back on the protocol i am using to take the biomass samples for the RNA extraction. The results should be used for a comparative differentially expression analysis among 4 different conditions in triplicates - i.e. 12 fermentations that has to be sampled at the same timepoint.
These are the sampling steps:
- Take 2 ml of sample
- Spin down cell pellet (5 min, in non-cooled centrifuge)
- Remove supernatant and immediately resuspend pellet in 5 ml RNAlater
- Incubate samples at 4degreesC for one hour
- Snap freeze samples in liquid Nitrogen
- Store at -80C until RNA extraction
What i am worried about is firstly, that the centrifuge i am using does not support cooling. I can compensate this by prechilling the sample tubes on ice before usage, but it will still get warm in the centrifuge. Can 5 min. of centrifugation affect the RNA expression e.g. by upregulation of heat shock proteins? Secondly, it takes ~ 10 minutes to resuspend all the samples in the RNAlater. Would this make a difference among the samples if i keep them on ice? Say one sample is immediately resuspended in RNAlater after centrifugation, and another is resuspended 10 min after.
What do you think of my sampling protocol? Do you think it will give me comparative samples?
Thank you!
I am going to do a transcriptome analysis of small scale S. cerevisiae fermentations using RNA-seq. I would like to get some feed-back on the protocol i am using to take the biomass samples for the RNA extraction. The results should be used for a comparative differentially expression analysis among 4 different conditions in triplicates - i.e. 12 fermentations that has to be sampled at the same timepoint.
These are the sampling steps:
- Take 2 ml of sample
- Spin down cell pellet (5 min, in non-cooled centrifuge)
- Remove supernatant and immediately resuspend pellet in 5 ml RNAlater
- Incubate samples at 4degreesC for one hour
- Snap freeze samples in liquid Nitrogen
- Store at -80C until RNA extraction
What i am worried about is firstly, that the centrifuge i am using does not support cooling. I can compensate this by prechilling the sample tubes on ice before usage, but it will still get warm in the centrifuge. Can 5 min. of centrifugation affect the RNA expression e.g. by upregulation of heat shock proteins? Secondly, it takes ~ 10 minutes to resuspend all the samples in the RNAlater. Would this make a difference among the samples if i keep them on ice? Say one sample is immediately resuspended in RNAlater after centrifugation, and another is resuspended 10 min after.
What do you think of my sampling protocol? Do you think it will give me comparative samples?
Thank you!