When doing gene expression analysis, that is, when determining the pattern of genes expressed at the level of genetic transcription, in a specific cell or under specific circumstances, quantitative primer design (qPCR) is certainly a crucial step.
Studying the formation of a gene product from its genetic code is a sensitive, and yet an important, process. Click this to find out more about what it entails.
Going beyond the static information of a genome, this analysis delves into the dynamic view of an organism’s biology.
Therefore, it is widely used in clinical and pharmaceutical settings and research, aiming at providing better understanding of gene pathways, individual genes, or greater gene activity profiles.
While there are various means of achieving this analysis, real-time PCR has become the most popular option.
Given the importance of such analyses, taking your time to design great qPCR primers is a must. Since the primers are fairly cheap, testing different pairs, aiming at finally choosing the right ones for you is a good thing to do.
Yet, since primer testing takes time, you’ll want to get your primers working as soon as possible. So, let’s learn how to design the primers.
Get a Primer Design Program
For a successful primer designing process, you’ll have to find the right primer design program for you. It is important for the program to be provided by a reliable source, so that you can be sure of its accuracy and efficiency.
PCR suppliers usually offer their programs, but it is eventually up to you to decide which one to use. Quality should always be your main concern, because you want the analysis to be successful.
Read more on primer design here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702850/
Get the Nucleotide Sequence
The entire process begins with getting the actual nucleotide sequence of the gene you’re interested in. Once you’ve selected the program, search its database and use filters to make the search easier.
Naturally, the actual search procedure will differ from one tool or another, depending on the actual dashboard, but it will all be pretty straightforward regardless of the program you choose.
After finding the necessary gene, click on it to find the nucleotide sequence reference. If the gene has different isoforms, there could be numerous sequences. So, remember to click on that specific isoform you’re actually interested in.
Once you do that, the next thing to do is click on a “pick the primer” button, or a link, depending on the design of the tool.
Input the Necessary Parameters
Having come this far, you’ll now have to carefully select the parameters that will be required by the tool. Most great tools will guide you through all of the options and explain what to input for each of the parameters, as well as why.
Basically, at this stage, you’ll have to set the settings for the primers, including the melting temperature, as well as the PCR product size. Some programs will also allow you to include the sequences for forward or reverse primers.
Double Check Everything
As designing primers is a delicate process and a rather sensitive one, since your analysis won’t work if not done correctly, make sure to double check everything before going any further.
Go through all of the parameters once again, and even check if you’ve chosen the correct sequence if you’re not sure. It’s always better to be safe than sorry, and since you have the right tool on your hands, make sure to use it correctly, so as to successfully complete the design.
Check the Output Page
Sure you’ve inputted all the information correctly? Then proceed to the output page. This is the page you want.
The page you’ve actually been after this whole time, because it provides you with a list of possible reverse and forward primer sequences you can choose from. Be patient, as this may take a while.
So, for effective qPCR primer designing, you’ll need to find a great, high quality tool first. Things will be easier from there, as you’ll simply have to follow the steps required by the program, input the necessary parameters and ultimately get your list of potential solutions.
Of course, always remember to double check everything.
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