how to do RT-PCR for16srRNA of a certain bacteria? - (Mar/25/2012 )

Hi! I am new in RT-PCR, and hope to get some advice in the forum.

I want to compare a G(+) bacteria in liquid sample between healthy and patients. I decided to compare the level of 16srRNA of this bacteria by RT-PCR. I have several questions below:


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 bacteria DNA?

Thanks a lot for any of your suggestions!

hi! i work with environmental bacterias, not with patogens, but i think you should be careful because 16S gene is a universal gene for all bacterias so you have to be sure that you don't have any other bacterias in your samples.
The primers are easy to find in many articles.
If you are to use the primers for 16S i think it will by no problem if you have DNA from eukariotes in your extract because 16 s is a very specific gene for bacterias. The problem will be with other bacterial DNA.

Thanks! My sample is whole mixture of bacteria. Is it very hard in this situation that I use 16srRNA to test just one specific bacteria? If so, maybe I should find the specific proteins especially expressed in my bacteria, and design primers on this protein?

Thanks!

Iulia on Thu Apr 12 12:08:12 2012 said:

I just did a similar thing with water enviornmental samples. We had to do both, campylobacter and ecoli. Both bacteria contain the 16s gene. YES, you need to specify primers because you might amplify something you didnt want to. We had searched and searched, designed, and destroyed many primers before we got the primers we use now, which do not cross amplify between camp or ecoli. 16s is a great start, but searching for updated articles (2010-Present) I think is your best bet.

Thanks a lot!

dp4616 on Thu Apr 12 15:24:35 2012 said: