I want to study the quantitative gene expression by real time PCR. For this i need to isolate total RNA and then do cDNA synthesis. I'm little confused that we should harvest the bacterial cells for RNA at primary culture stage or secondary culture stage after induction with 1mM IPTG. The BL21 DE3 bacterial cells are transformed with the recombinant construct. Does 1mM IPTG in 50ml or 100ml of LB interfere with the isolation or extraction process?
IPTG will not interfere with RNA isolation. Now, when when to take the sample depends on what you want to study. Usually (especially when using quantification by qPCR instead of e.g. deep sequencing) people are interested in changes in gene expression -or lack thereof- between at least two experimental conditions, so presumably you should take at least 2 different samples. What experimental conditions do you want to compare?
Dear Anam...
Being more specific will be very helpful in giving you a specific answer from experts.
I'm assuming your gene of interest is under the control of an inducible promotor and that's why you would add the IPTG. If that's the case, then you should collect your cells after you have added IPTG for at least one of your time points.
But it really sounds like you need to talk to your advisor first. You should plan out your experiment (collection time points, number of samples, design your primers, etc.) BEFORE you start growing your cells. The time to collect your cells should not be a guess.
Absolutely depends on your experimental design, you can isolate total RNA from any point, fresh culture is preferred, during stationary and late stationary you will get more degraded low quality RNA, but as per your experimental demand you can extract from any time point. If you are going to perform relative quantification it will be fine.IPTG does not interfere with RNA isolation. You can perform as wash step before lysing cells.
Thanks
Thanks all for your suggestions. My genes of interest is under IPTG inducible promoter. My supervisor has advised to do study the difference in the level of mRNA transcripts of both the genes through qPCR then in this case what should we do????
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