Dear,
I am working with a slow growing bacteria.
I need to harvest 10 ml at the low OD and approximately 5ml at high OD for RNA isolation and transcriptomics.
I saw that most people use RNA protect from Qiagen, but in my case the volume is too big for all my samples.
My questions are:
1. RNAlater should be added after spinning the culture, 5-10 volume of the pellet? right
2. Can RNAlater be used for bacterial culture? I saw that mostly used with tissues
3. Does the transcription patter change in these 5 min of centrifugation prior to the addition of the RNAlate?
Best,
Hi Miriam,
We use RNALater for our bacterial culture and keep them on the bench for up to 1 day or at 4*C for 1 week and extract RNA with high yields and good downstream applications. We usually harvest 3-5 mL of bacterial culture and centrifuge at high speed for 2 m, then resuspend in 750 ul RNALater (this process may depend on your bacteria?). When we are ready to extract the RNA, I add my lysis buffer directly to the RNALater + bacteria mixture. I have not noticed any transcriptional change by doing it this way. Hope this helps!
Hi Miriam,
We use RNALater for our bacterial culture and keep them on the bench for up to 1 day or at 4*C for 1 week and extract RNA with high yields and good downstream applications. We usually harvest 3-5 mL of bacterial culture and centrifuge at high speed for 2 m, then resuspend in 750 ul RNALater (this process may depend on your bacteria?). When we are ready to extract the RNA, I add my lysis buffer directly to the RNALater + bacteria mixture. I have not noticed any transcriptional change by doing it this way. Hope this helps!
Thank you for you help :) as you have a good experince in doing this you may help with some more points
1. High speed of sentrifugation for 2 min is it enought with 13000 rpm or this is too high?
2. after adding the RNAlater to the pellet, as the manufacturer¨s recomndation says that i have to leave the sample ON at 4C and then freez at -80C is this safe? or it is better to freez imidiatly at -80 after resuspending the pellet in RNAlater?
Thank you for mentioning the RNA extraction after the RNAlater:) so it is safe to proceed with the lysis step without removing the RNAlater in advance.
Best,
Miriam
Dear Miriam, you can follow the procedure described by Meghan. It is the best way.
Hi Miriam,
We centrifuge at 14800 rpm for 2 minutes to get a pellet and everything seems fine.
I have always stored samples ON at 4C and then frozen, I do not have experience with freezing immediately at -80C.
Good luck!
Dear Meghan,
Can you please tell me what kind of bacteria did you use? I am trying to extract total RNA from M. smeg collected on swabs. As per your recommendation, I plan to use around 750uL of RNA later solution as my swab collection buffer and freeze them. Once ready for extraction, I will throw in lysozyme into the tube directly to extract total RNA.
Appreciate your help!
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