PCR for gene expression [closed]

Question

Could somebody please explain the basic principle behind how qPCR can detect if a gene is expressed or not? I tried looking at literature on ScienceDirect and other websites but I could not find any information. I understand what qPCR is supposed to do - amplify a target DNA and provide numbers for the resulting DNA after cycles. I am familiar with the concept of gene expression.

Answer 1

What is qPCR? What's the central concept?

qPCR (or more stringently, qRT-PCR, a term that is less ambiguous) usually refers experiments which use quantitative polymerase chain reactions to quantitate (measure) gene expression in real time ("RT"), and when the source material is RNA, it is reverse-transcribed first ("RT") because PCR does not work well with single-stranded nucleic acids such as RNA.

The idea behind it is simply two-fold:

• we amplify DNA (using PCR), and
• we measure the DNA in real time during each step/cycle of the PCR, until the reaction is exhausted or plateaus or is sufficient for making a determination about DNA amount.

Over many PCR cycles, say 40 or 50, the amount of DNA product reaches a plateau that is not directly correlated with the amount of target DNA initially. So we must survey the amount of DNA before we hit the plateau! That's why we do it in real time. Simple.

How is DNA actually measured?

We measure DNA at the end of each PCR cycle using reporters. Typically, a fluorescent dye that intercalates (binds) with DNA is used. To simplify somewhat, more fluorescence means more DNA strand. We can measure absolutely (comparing to known amounts, or DNA standards, which we calibrate to) or relatively (relative to another gene or segment of DNA which we are amplifying using known primers). When you do quantitation, you must make sure as best as possible that your sample and the standard have the same amplification efficiency, so that the assumption that each cycle will amplify equally across samples will be assured.

How does qPCR measure gene expression specifically? DNA is not gene expression.

We use the amount of DNA transcript (mRNA) as a proxy for the expression of the gene. We can treat expression as numerical quantities. For example, 5 mRNA strands that act as transcripts of the insulin INS gene are assumed to be a tenth of what 50 insulin mRNA transcripts are. In the second case, you have a 10-fold higher expression than in the case with only 5 transcripts.

However, qPCR does not work with RNA (single-stranded nucleic acids) since PCR depends on two-strandedness of DNA. So to quantitate a gene's expression (mRNA) we must first reverse transcribe all the mRNA into complementary DNA (cDNA) which we can then put into the qPCR machine!

So, the proxy for gene expression is mRNA amount, but now we are using a proxy for mRNA amount in the form of cDNA.

With this cDNA, we can now precisely quantify how much of it there is. Then we can make an indirect statement about how much mRNA for specific genes we found in our sample (e.g. biopsy, lysed cells, bodily fluids, harvested tissues, etc.). So we've achieved quantifying expression of our sample.

The devil in the details

Importantly, the whole process takes place within PCR conditions, so you are very dependent on your primers and thermal cycling conditions. If your extension temperature step is too short, long strands may not have enough time to complete duplication by the polymerase. If your primers are designed incorrectly, or are in conditions in which they cannot anneal efficiently, you will have incorrect amplification and the quantitation will not be possible. If you do not prepare the initial reaction with sufficient dNTPs (DNA building blocks), your reaction will be exhausted prematurely. If you forget to add a polymerase enzyme, there will be no amplification. For some reactions, you need to use specific polymerase enzymes which are more thermostable, more efficient, or are more stringent in that they make fewer errors in duplicating DNA strands. A lot of little things must be satisfied in order to run a good qRT-PCR with dependable read-out, so please refer to standardized practices and considerations. Here is an old but good one to familiarize yourself with.