2 replies to this topic

[Annice]

Hi all,

I'm preparing for my study which use relative qRT-PCR to determine chnages of mRNA level of a target gene on treated bacteria culture. I'm new to qRT-PCR and this is the first time I work on it. I'm quite worry about it. I was wondering

1. is it fine to use 16S as housekeeping gene?
2. 1 step or 2 step qRT-PCR?

Would you give me some advices, please?

Thanks so much.

[littleaxt]

Heiho!

1. If it's fine depends on your experiment. Test if your treatment does not effect your reference gene or search for literature if someone else did test it already. Also it is often recommended to use more then one reference gene and calculate a normalization factor.
2. I prefer two step, because it allows replicates of each reverse transcription reaction in your real-time PCR, and therefore you have more control over the experiment.

all the best

jan

Annice, on Apr 19 2009, 03:54 PM, said:

Hi all,

I'm preparing for my study which use relative qRT-PCR to determine chnages of mRNA level of a target gene on treated bacteria culture. I'm new to qRT-PCR and this is the first time I work on it. I'm quite worry about it. I was wondering

1. is it fine to use 16S as housekeeping gene?
2. 1 step or 2 step qRT-PCR?

Would you give me some advices, please?

Thanks so much.

[HomeBrew]

Check to see how many copies of the 16s RNA gene there are in your bug-- in the bacteria I work on, there are six identical copies scattered throughout the genome.  Since I assume there's only one copy per chromosome of your target gene, the 16s RNA gene is probably not be the best housekeeping gene to use as a reference.