real-time PCR?

[w...b]

so i have an essay on this subject and i cant seem to grasp the concept of PCR. i understand that its an amplification technique, and real-time PCR has higher quantification compared to conventional PCR.

 

but what information do we get. im reading these research articles and i still don't understand what the information is used for. for example one article is looking at the bacteria genus Lactobacillus in infant feces. they determined that an infant that gets breast fed and an infant that gets fed a sugar combination(galacto-oligosaccharides, fructo-oligosaccharides) has the same amount of Lactobacillus. they used real time PCR to get this information. but i dont understand how using the amplification of the DNA or complementaryDNA was used.

 

is it because by running different samples for the same amount of cycles they are able to compare the two. thus getting data to determine the % of this bacteria in the feces?

 

i know this is off topic to this forum but i need the help.

 

thanks

 

 

w...b

[SGMD09]

Real time PCR involves amplification of DNA in a machine that measures the concentration of new DNA in real time. By generating a standard curve using DNA of known concentrations, you can determine how much DNA was in your original sample. It's commonly used as a specialized form of RT-PCR, in order to quantify mRNA/gene expression.

[AMmd]

Real time PCR involves amplification of DNA in a machine that measures the concentration of new DNA in real time. By generating a standard curve using DNA of known concentrations, you can determine how much DNA was in your original sample. It's commonly used as a specialized form of RT-PCR, in order to quantify mRNA/gene expression.

 

Actually you use mRNA that you converte to cDNA (complementary DNA) by the RT enzyme (reverse trascripatase). which will be amplified in the pcr. This helps you to compare two or more samples for the amount of original mRNA product...

 

you cant really say that you have the same amount of bacteria in two samples... what you can say, is that the gene you are looking for (or portion of mRNA) is expressed in the same amount in both samples. This could be mislading, in the sense that you could have a smaller amount of bacteria in one sample but the genes you are looking for are up regulated in that same sample, which gives you the same results in the real time pcr. However, this is counteracted by comparing the targeted gene with a house keeping gene, such as actin, which is expressed in all cell at a similar amounts. Problem with bacteria, is thay dont have actin. so perhaps MreB.

hope this helps

good luck

[haiku_guy]

You still have a "housekeeping" gene for normalization when using the std curve method.

 

You end up with two curves. You use them to then get realative amounts for the unknown gene of interest and the housekeeping gene.

 

then you can use the ratio of unkown:housekeeping to determine relative changes in the expression of that gene.

 

I have a detailed protocol for this if you want it email/PM me and we can go over the sailent points.

 

P.S. I found the level of actin in the cell can fluctuate. I used GAPDH as my housekeeping gene for normalization

 

P.P.S. You don't need to do the amplification in a real-time machine if you use the TAQ-man system. As it is based on FRET technology, you can take a starting flouresence reading and an endpoint reading and do the cycling in a normal pcr machine.