sample storage method for extracting 16s rRNA - (Nov/15/2011 )

Hi, I want to check microbe 16srRNA from human sample (liquid). I want to know if it is propriate to store the samples (liquid) at -80C for months, and extract DNA later?

Are there any special needs? For instance, adding agents to stabilize DNA?

Thanks a lot!

It should be fine to store at -80, RNA is pretty stable at that temperature, and if it is wrapped up in the ribosome, it is even more stable.

And you probably will really extract DNA rather than RNA, and then amplify the 16s rRNA fragment of the genomic DNA.

I think yes i agree with you.

Hi! Thanks a lot for your responses. I have just got the sample.

1. which general procedures should I go through? I think it includes: DNA extraction-->get primers for the specific 16srRNA--> do common PCR to check if the primers are good-->do RT-PCR (include positive and negative control)-->data analysis. Is that correct?

2. The sample contains mammal cells and bacteria (gram- and grm+). I am interested in a G+ bacteria 16srRNA. As far as I know, the G+ bacteria DNA are harder to extract. How should I extract the DNA?

3. How should I design the primers for 16srRNA? Which database is good for designing the specific primer?

4. If the extracted DNA contains mammal cells DNA, and bacteria DNA, how should I quantify the DNA to make sure I load the same amount of bacteria DNA?

Thanks a lot! Look forward to your responses!