Ok, for qPCR, using cDNA prepared from extracted RNA, you are definitely, definitely going to want to design primers that are NOT in introns.

Intronic primers would potentially allow you to identify immature unspliced transcripts, but these are transient at best and thus hard to quantify, unless they are your specific research focus.

I am going to assume you are instead interested in mature transcripts, so for this my typical approach would be to go to ensembl and find my gene in my species of interest, then export the transcript sequence as a text file. Ensembl usually exports not only the whole transcript but also the individual introns/exons that comprise it.

Look at the transcript in ensembl and see if there are any really big introns (more than a kilobase is enough, but 10k or more is ideal).

Now find the exons either side of that intron in your exported data, and paste them into primer3:

https://primer3.ut.ee/

Primer3 (or primer3plus, the latest iteration) is a free software package pretty much specifically for designing qPCR primers, and it's great.

Assuming, say, we had exons 3 and 4, paste them in as follows

First exon 3:

Exon 3 sequence here...ATGGCTGGCTA

Now add a square bracket before the last two nucleotides

Exon 3 sequence here...ATGGCTGGC[TA

Now paste exon 4 sequence immediately after (I've put it in bold)

Exon 3 sequence here...ATGGCTGGC[TAATTCGGTCATG....Exon 4 sequence

And again, add a square bracket

Exon 3 sequence here...ATGGCTGGC[TAAT]TCGGTCATG....Exon 4 sequence

Then hit "design primers". What the square brackets do is tell it "give me primers that specifically lie either side of these four nucleotides", so that your amplicon will definitely span the exon:exon junction.

The idea here is that unspliced transcripts (or more importantly, genomic DNA) will contain the 10kb+ intron, and these two primer binding sites will thus be separated by an enormous amount of sequence, while in the mature transcript, they will be very close together. PCR will thus ONLY amplify your mature transcript sequence, which is what you want. If you can, therefore, always design primers that lie in exons either side of a large intron.

Primer3 usually gives you a top scoring pair, but also five or six alternative options: I would pick at least three pairs and try all of them, keep the best. Primers are cheap, and it's a lot easier to start with a qPCR that is really, really trustworthy than it is to spend days trying to optimise a specific PCR reaction.

As far as product length goes, again, Primer3 will sort this out for you, but as a rule of thumb, anything from 80-200 bases or so is normal for qPCR. Long enough such that it can easily be distinguished from primer dimers, but nice a short so you can cycle rapidly.

Ok, for qPCR, using cDNA prepared from extracted RNA, you are definitely, definitely going to want to design primers that are NOT in introns.

Intronic primers would potentially allow you to identify immature unspliced transcripts, but these are transient at best and thus hard to quantify, unless they are your specific research focus.

I am going to assume you are instead interested in mature transcripts, so for this my typical approach would be to go to ensembl and find my gene in my species of interest, then export the transcript sequence as a text file. Ensembl usually exports not only the whole transcript but also the individual introns/exons that comprise it.

Look at the transcript in ensembl and see if there are any really big introns (more than a kilobase is enough, but 10k or more is ideal).

Now find the exons either side of that intron in your exported data, and paste them into primer3:

https://primer3.ut.ee/

Primer3 (or primer3plus, the latest iteration) is a free software package pretty much specifically for designing qPCR primers, and it's great.

Assuming, say, we had exons 3 and 4, paste them in as follows

First exon 3:

Exon 3 sequence here...ATGGCTGGCTA

Now add a square bracket before the last two nucleotides

Exon 3 sequence here...ATGGCTGGC[TA

Now paste exon 4 sequence immediately after (I've put it in bold)

Exon 3 sequence here...ATGGCTGGC[TAATTCGGTCATG....Exon 4 sequence

And again, add a square bracket

Exon 3 sequence here...ATGGCTGGC[TAAT]TCGGTCATG....Exon 4 sequence

Then hit "design primers". What the square brackets do is tell it "give me primers that specifically lie either side of these four nucleotides", so that your amplicon will definitely span the exon:exon junction.

The idea here is that unspliced transcripts (or more importantly, genomic DNA) will contain the 10kb+ intron, and these two primer binding sites will thus be separated by an enormous amount of sequence, while in the mature transcript, they will be very close together. PCR will thus ONLY amplify your mature transcript sequence, which is what you want. If you can, therefore, always design primers that lie in exons either side of a large intron.

Primer3 usually gives you a top scoring pair, but also five or six alternative options: I would pick at least three pairs and try all of them, keep the best. Primers are cheap, and it's a lot easier to start with a qPCR that is really, really trustworthy than it is to spend days trying to optimise a specific PCR reaction.

As far as product length goes, again, Primer3 will sort this out for you, but as a rule of thumb, anything from 80-200 bases or so is normal for qPCR. Long enough such that it can easily be distinguished from primer dimers, but nice a short so you can cycle rapidly.

Hello Eduardo. How are you doing today?

Yes, for qRT-PCR you need to design your primers in the exon region. Try to set the primers flanking a big intron. For example, the forward in the exon 3 and the reverse in the exon 4, the intron between those has at least 500bp. This will avoid genomic DNA amplification during the reaction. Polymerase enzyme present in the qRT-PCR mix does not amplified bigger fragments.

Primers for qRT-PCR your conventional PCR has around 20 to 25 bp. However usually the qRT-PCR amplicons have around 70 to 150bp, very small fragments compared to conventional PCR, (500 to 1000bp). The melting temperature depend of the percentage of G-C content in your primer. Usually we design primers at 60°C Tm for standard.

Well, my best regards and good luck in your research.

The following paper would help:

Validation of a primer optimization matrix to improve the performance of reverse transcription–quantitative real-time PCR assays

The following paper would help:

Validation of a primer optimization matrix to improve the performance of reverse transcription–quantitative real-time PCR assays

For gene expression studies always design primers which span exon-exon junctions for amplification of cDNA. If you design your primers for exons you might end amplifying genomic DNA because a small amount genomic DNA might be present in your RNA prep. Also avoid regions with SNPs.

Thank you very much to all of you. All of these clarifications were important.