qPCR acronym as quantitative PCR is a technique to measure the amount of DNA present in a sample. It can study gene expression by utilizing either the TaqMan probe or SYBR green dye.
Quantitative PCR or RT-PCR is one of the most popular and useful variations of PCR. Conventionally, the PCR is a polymerase chain reaction that amplifies the DNA.
qPCR has been modified to not only amplify the DNA but also to quantify it, meaning, it can measure the amount of DNA/RNA or the number of DNA/RNA templates present in a sample.
Henceforth, it has a wide range of applications in gene expression studies and microbe quantification. In addition, scientists are using it for various genetic studies and validation assays.
The qPCR assay relies on two types of chemistries; probe-based assay and dye-based assay uses TaqMan probe and SYBR green dye, respectively. Both have different applications and utilities, however, the TaqMan probe-based qPCR assays have greater accuracy so, are highly recommended.
Let’s come directly to the point if you are landing on this article, meaning, you have substantial knowledge regarding the qPCR or RT PCR (Real-time PCR) and you want a direct protocol.
I have written this article to provide you a general protocol for qPCR which you can utilize to design your own protocol for various assays. In this article, I have enlisted the required chemicals, utilities, instruments and other things. I also have given the quantity and requirement of different reagents used.
So the present article will help you a lot in your PCR journey. Stay tuned.
Key Topics:
qPCR protocol workflow:
The whole process of qPCR can be divided into 4 steps; Preparing cDNA, preparing reaction mixture, Running the qPCR assay and Analyzing results. Let’s first discuss each step elaboratively and then the protocol.
Preparing the cDNA sample:
When we perform a qPCR assay for mRNA quantification or gene expression studies we first need to prepare the cDNA template. It’s an acronym as complementary DNA to our transcript.
If you don’t know what the transcript is! You can read our previous article: What is transcriptomic?
The target mRNA to study is our transcript. So basically, we need only a cDNA and not “all DNA” and for that we need to first perform the reverse transcription PCR. Here the enzyme reverse transcriptase converts the mRNA into the cDNA.
Nowadays, high-quality cDNA amplification kits are available with the ready-to-use reverse transcriptase enzyme. So no need to do things manually.
Preparing the qPCR reaction:
Once we get the desired amount of cDNA, we can use it to prepare the qPCR reaction. It’s a cocktail or combination of chemicals and reagents that amplifies our cDNA.
It usually consists of the master mix, primer set, probe or dye, cDNA and nuclease-grade water. Noteworthy, reaction preparation is a key step in qPCR protocol to achieve good results.
Some reactions known as a single-step RT-PCR performs the reverse transcription and qPCR in a single reaction, however, are less effective and efficient. Two-step reactions are usually recommended for achieving specificity and storing the cDNA for future use.
Running the qPCR protocol:
The next step is running the protocol on a thermocycler. Denaturation, annealing and extension are three important PCR steps while the run duration and steps may vary depending upon assay requirements.
For instance, sometimes the annealing and extension steps are combined in order to deactivate the reverse transcriptase and achieve specific probe and primer binding (to the template).
Different assays and reactions need different PCR setup, reaction time and cycle properties. Nowadays kit manufacturers provide their own standardized protocol.
Evaluating results:
As per the requirement of the assay results are evaluated. Several ways to interpret results are standard curve analysis, Ct value analysis, relative quantification and absolute quantification.
Move back to our previous article on the real-time PCR which included all the explanations related to results and interpretations. The link is given above the fold of the article, or you can read it here: Real-time PCR.
Requirements:
qPCR mastermix, probe mix or dye mix, Reverse transcription reagent kit, Other PCR reagents, PCR machine, Reaction plates and other accessories, centrifuge machine, Microcentrifuge, Pipette & Pipette tips, vortexer, nuclease-free water and template.
qPCR protocol:
Preparing the cDNA:
We are discussing the protocol for mRNA quantification using the reverse transcription mechanism.
Extract RNA from a sample using either manufacturer’s protocol or ready-to-use RNA isolation kit. Use 1μg of 20 μl pure RNA to prepare cDNA.
After isolating the RNA, quantify and measure the quantity of the RNA. Only pure and good-quality RNA should be used in the reaction.
qPCR reaction setup:
Different reagents used in the reaction preparation are given in the chart below.
| Reagent | Concentration | Quantity |
| 20mM dNTPs | 2mM | 2 μL |
| 0.1 mM DTT | 0.08 mM | 4 μL |
| Random Hexamer primers (500 ng/μL) | 0.5 ng | 1 μL |
| 10X cDNA synthesis buffer | 10X | 2 μL |
| MgCl2 (If required) | 2mM | 4 μL |
| Oligo (dT) primer | 2.5 μM | 1 μL |
| RNase inhibitor | 20 U | 0.5 μL |
| Reverse transcriptase enzyme | 10 U | 0.5 μL |
| RNA sample | 1 μg | 10 μL |
| Nuclease free water | As per requirement |
Note that it isn’t a reaction cocktail, it’s a list of reagents, concentrations and quantities required. The recipe may vary depending upon the manufacturer. For example, some have given enzymes in the reaction buffer while some reactions do not need MgCl2.
The ideal reaction mixture is given in the table below,
| Reagent | 20 μL reaction | Concentration |
| dNTP mix | 1 μL | 10 μM each |
| Oligo dt Primers | 1 μL | 0.5 mM |
| RNA sample | 10 μL | 5 μg |
| >>>>>>>>>>>>>>>> | >>>>>>>>>>>>>> | >>>>>>>>> |
| 5X first strand buffer | 4 μL | 5 X |
| 0.1 M DTT | 2 μL | 50.1 M |
| Reverse transcriptase | 1 μL | 200U |
| RNA inhibitor | 1 μL | 40 U |
| ************ | ******************** | ********** |
>>>>>>> incubate the sample at 65 ºC for 5 min.
******** incubate the sample at 42 ºC for 50 min. Immediately after, denature the sample at 70 ºC for 15 minutes. Store the sample at -20ºC for further use.
After preparing the reverse transcription reaction, we have to perform a PCR in order to convert our mRNA to cDNA. Here is the reaction setup for that. The reaction is completed in two steps, all the preparation steps are explained here:
- Add 10 μL each dNTP (10 μM) to the reaction tubes first.
- Now add 1μL the oligo (dT) primers (0.5 mM) to the reaction.
- Add 10 μL RNA template sample to the reaction and heat it at 65ºC temperature for 5 minutes.
- Now add 4μL first-strand RNA synthesis buffer. A buffer typically consists of components that are needed to boost the reaction.
- Now add 2 μL of 0.1M DTT to the reaction.
- In the last step add 1 μL of reverse transcriptase enzyme and RNA inhibitor to the reaction and complete reaction with the RNase-free water.
- Incubate the sample at 42ºC temperature to stop enzyme activity and either store it or send it for qPCR.
Preparing qPCR:
| Reagent | 30 μL reaction | Concentration |
| dNTPs | 1 μL | 10 μM each |
| Forward primer | 0.5 μL | 10μM |
| Reverse primer | 0.5 μL | 10μM |
| SYBR green dye mixture | 15 μL | 1X |
| cDNA | 5 μL | 250 ng |
| RNase-free water | Up to 30 μL | |
| Final concentration | 30 μL |
Preparing qPCR reaction using TaqMan
| Reagent | 30 μL reaction | Concentration |
| dNTPs | 1 μL | 10 μM each |
| Forward primer | 0.5 μL | 10μM |
| Reverse primer | 0.5 μL | 10μM |
| TaqMan probe buffer | As per requirement | |
| TaqMan probe | 1 μL | 1X |
| cDNA | 5 μL | 250 ng |
| RNase-free water | Up to 30 μL | |
| Final concentration | 30 μL |
PCR reaction set up:
| Cycle 1 | Cycle 2 to 40 | Cycle 2 to 40 | |
| Step 1 | Step 1 | Step 2 | |
| Temperature | 95°C | 95°C | 60°C |
| Time | 3 to 5 minutes | 30 sec | 1 minute |
Once the cDNA is synthesized we can perform the rest of the qPCR steps as followed,
Add 0.5 μL each forward and reverse (10μM) primer to the reaction.
Add 15 μL of 1X SYBR green dye mix to the reaction.
Add 5 μL of cDNA to the reaction having a concentration of 250 ng.
Add RNase-free water in the final step to make the final reaction 30 μl. We can also prepare a 20μL PCR reaction if required.
After completing reaction preparation, mix all reagents well using vertex mixing and put them in the machine.
Set up the first PCR cycle at 95ºC for 3 to 5 minutes.
Annealing and extension are done at above 60ºC temperature for 30 sec to 60 sec.
Perform 40 cycles for qPCR amplification.
So this is an ideal protocol for the cDNA synthesis using the reverse transcription PCR followed by the qPCR protocol.
To make things easier for you I am giving you a standard microbial identification protocol so that you can understand how qPCR protocols vary from assay to assay.
So here is a protocol for (n) microbial detection using the single-step qPCR reaction.
In the first step, the RNA is isolated from the sample, purified, quantified and processed for reaction preparation. The table for reaction preparation is given below,
| Reagent | Concentration |
| 10X RT-PCR buffer | 12.5µL |
| Reverse transcriptase enzyme | 1µL |
| Forward primer | 0.4µL |
| Reverse primer | 0.4µL |
| TaqManProbe mixture | 0.4µL |
| Nuclease free water | 5.3µL |
| RNA sample | 5µL |
| Total reaction volume | 25µL |
After preparing the reaction, the PCR cycling conditions are set as per the table given below.
| Pre holding | 60ºC | 30 sec | |
| cDNA preparation | 50ºC | 30 min | |
| Inactivation of RT-Enzyme | 95ºC | 10 min | |
| DenaturationAnnealing and extension | 94ºC 55ºC | 15 sec 1 min | 45 PCR cycles |
| Post holding | 60ºC | 30 sec |
After completion of the run, the results are evaluated as per the requirement. To measure the infectivity, usually, the Ct value is taken into account.
Wrapping up:
qPCR protocols vary depending upon the requirement of the assay, however, ready-to-use kits have their own protocols and reagents so no need to worry about it. As a genetic student, one should know manual things.
Obviously, you can’t use this protocol directly in your lab but you get a direction on how the qPCR protocols are designed and prepared. And that’s why I have written this article, I hope this article may help you to “understand” how qPCR protocol works!
Sources:
Bookout AL, Mangelsdorf DJ. Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nucl Recept Signal. 2003;1:e012. doi:10.1621/nrs.01012.
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