RT-qPCR (Reverse Transcription/Real Time - quantitative Polymerase Chain Reaction) theory & practice

Quantitative PCR (qPCR) is a way to “count copies” of specific pieces of DNA - and when those specific pieces are DNA copies of RNA (like the RNA copies of protein recipes) we call it Reverse Transcription (RT) qPCR - RT can also stand for Real Time, which refers to the measuring - the products are detected as they’re made because of the fluorescence they cause (either because of generic dyes or specific probes) - unlike traditional PCR (Polymerase Chain Reaction) which you use just to make copies of defined stretches of DNA, with the goal of making as many copies as possible, RT-qPCR counts the copies as they’re made to figure out how many copies you started with.  
 
blog form (with full text & static graphics): http://bit.ly/rtrtqpcrprimer  
  
In the beginning there aren’t enough copies to measure directly, so you stabilize the recipe copies and make more copies of them to amplify the signal to a detectable level - the more copies you start with, the fewer amplification cycles you’ll need to do this - so you can compare how many cycles are required for different recipes, a number referred to as the quantification cycle (Cq) value - a lower Cq value means more recipes, and likely more protein.  
 
In PCR, we can use DNA Polymerase (DNA Pol) to copy DNA in little tubes. More here: http://bit.ly/pcrtrain     
  
We don’t want it to copy everything, but thankfully for us, we can utilize its limitations to our advantage to only copy defined stretches of DNA using primers.

So the first step in RT-qPCR (after you isolate the RNA) is making DNA copies of the RNA copies of the DNA recipes through REVERSE TRANSCRIPTION. Normal transcription goes DNA→ RNA. REVERSE transcription goes RNA→ DNA. It uses a different polymerase (instead of the usual DNA-RNA or DNA-DNA Pols you need an RNA-DNA Pol - we call such Pols reverse transcriptase) - and we call the DNA copies of the mature mRNAs complementary DNA (cDNA)  
  
The reverse transcriptase can make DNA copies of RNA, but it still has the limitation of needing a double-stranded starting platform - so you need to provide primers for it, such as oligo(dTs) or random hexamers (more details in link). After you make the cDNA copies of “everything” or at least all the mRNAs, you want to make copies ONLY of the recipe you’re interested in. So instead of aiming for genericness, you want your primers to be super specific. And you’ll need primerS now Because now we want to amplify - with reverse transcription we only made 1 strand of cDNA, but now we want to make lots of copies. So we need to make a second strand from that first strand and then we can use those strands as templates for the other strands so you can make more and more and more…  
  
Since you’re now just copying DNA to DNA, you can use a “normal” DNA Pol. And just like in normal PCR, qPCR is performed in cycles of temperature changes - MELT (heat up to separate strands)→ ANNEAL (cool down to let primers bind & Pol latch on)→ EXTEND (let Pol lay down complementary track)→ REPEAT.   
  
So you need 2 primers - one for each strand - one will define the start & the other the stop for the region you want to copy. The first primer will bind the cDNA (at where you want to start) and Pol will start copying it 5’ to 3’ until it falls off the end of the cDNA or it runs out of steam, etc. And then in the next cycle that second strand needs a primer that bind it - and where it binds will define the start of where that strand starts. And it’ll go to where the 1st primer started because that’s as far as the strand its copying goes. So from then on, your strands will be the same length, bookended by those primer sites.  
  
Each round of PCR, another copy can be made from each copy, so you increase exponentially. In the very beginning you can’t tell this though because the levels are so low you’re below the background & just see “noise.” But soon you’ll enter the exponential phase where you get measurable doubling each cycle - and since you start with way more supplies (primers, dNTPS, etc.) than you need, you don’t have to worry about running out. But later on you do start running out, so copy # stops growing exponentially, and your curve plateaus.   
  
If you can directly couple the amount of light given off to the number of copies you make, such as with Taqman probes, and you use a special PCR machine with a fluorescence detector, you can read out - in “Real Time” - the number of copies you’re making.  
 
You can plot cycle # vs fluorescence and - in either type of measuring - what you’re looking for is a value called the Cq value (quantification cycle) which is the # of cycles it takes to pass a “threshold line” corresponding to the background fluorescence level - the more copies you start with, the fewer cycles it will take (lower Cq) and the more “left-shifted” your curve will be.  

some good webinars:    • Видео   &
   • Видео