Dear Sulail

see attached

In essence, ignore any delta G values < -5kKal/Mol

In terms of structure, this usually translates to 3 or more consecutive G/C residues particularly within the last 5 bp at the 5' and 3' end of primers: Thus, don't worry about the putative structures predicted in your outputs. They are theoretical more than real 

Therefore ignore the following

1. Predicted hair pin loops where you have just 2 consecutive G/C residues

2. More than 2 G/C residues but with AT residues in between, i.e. not consecutive

3. 2-3 consecutive GC residues but in the middle of primers (where steric hinderance makes those structures difficult to form in the first place)

In summary, if delta G's are < -5-10 k Cal Mol, equating to internal consecutive GC residues or GC Residues separated by AT residues they are not thermodynamically stable and will thus contribute in any meaningful or steady state way to reducing the effective concentration of your primers; if they form at all they fall apart in Micro seconds 

Conversely structures with Delta G values > -10kCal/mol tend to have 4 or more consecutive GC residues are are located at the extreme end of primers: These are thermodynamically stable are thus form steady state structures in real time and therefore reduce the effective concentration of your primer pool and hence the efficiency of your PCR reactions

If structures are found to have delta G values > -10kCal/mol then your best option is ALWAYS to design new primers in another region avoiding 3 or more consecutive GC residues. If this isn't possible then you can

• add 1M betaine and/or 5% DMSO to your PCR or qPCR reaction 
• you can even add to the initial RT reaction or to 1 step RT qPCR reactions

I hope this helps !!

These are both for Acta2. 

delta G for hairpin less than -3 and for hetero and homo dimer around -6 is fine but dimers engaging 3' end of primers are not favourable.

I used Netprimer with the graphical output and sometimes I inserted mismatch bases to get rid of dimers/cross dimers/hairpins. Be aware of the wrong Melt temp. To get an idea of the real Melt temp, substract 3°C for each mismatch G/C, and -2°C for each mismatching A/T. However, the net primer java version does not work any longer for me ... so I decided to use https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html

However, there is no info about hairpins. I do only use primers with no hairpins, dimers, cross dimers int the old net primer version, respective no hairpins, dimers, crossdimers in the most sensitive Thermo app.

Cheers.

Dear Sulail

see attached

In essence, ignore any delta G values < -5kKal/Mol

In terms of structure, this usually translates to 3 or more consecutive G/C residues particularly within the last 5 bp at the 5' and 3' end of primers: Thus, don't worry about the putative structures predicted in your outputs. They are theoretical more than real 

Therefore ignore the following

1. Predicted hair pin loops where you have just 2 consecutive G/C residues

2. More than 2 G/C residues but with AT residues in between, i.e. not consecutive

3. 2-3 consecutive GC residues but in the middle of primers (where steric hinderance makes those structures difficult to form in the first place)

In summary, if delta G's are < -5-10 k Cal Mol, equating to internal consecutive GC residues or GC Residues separated by AT residues they are not thermodynamically stable and will thus contribute in any meaningful or steady state way to reducing the effective concentration of your primers; if they form at all they fall apart in Micro seconds 

Conversely structures with Delta G values > -10kCal/mol tend to have 4 or more consecutive GC residues are are located at the extreme end of primers: These are thermodynamically stable are thus form steady state structures in real time and therefore reduce the effective concentration of your primer pool and hence the efficiency of your PCR reactions

If structures are found to have delta G values > -10kCal/mol then your best option is ALWAYS to design new primers in another region avoiding 3 or more consecutive GC residues. If this isn't possible then you can

• add 1M betaine and/or 5% DMSO to your PCR or qPCR reaction 
• you can even add to the initial RT reaction or to 1 step RT qPCR reactions

I hope this helps !!

Many thanks to all of you for your valuable answers!

Many of these thermodynamic and Tm-related algorithms are still evolving.  After a certain point, nothing beats empirical testing of different primer pairs. Some primer pairs that shouldn't work at all actually work in many situations; inexplicably.

Reduce Mg++ concentration. The sequences are OK.

I need a little advice. I designed a primer pair. The forward primer has a primer binding site somewhere at the mid EXON 9. Then, there is an intron (more than 1000 bp) followed by a short exon (EXON 10 having 57 bp). Next, there is a short intron (3 bp).. Now comes the primer binding site for reverse primer which is at the end of EXON 11 & beginning of EXON 12. Exon 11 & 12 are separated by an intron of 411 bp. Can someone comment on the primer design, please.

Dear Fatima

• for RT PCR where you substrate is cDNA without introns the presence of introns is therefore of no consequence to PCR amplification from your RT (cDNA) product
• Indeed one of your primers should be exon spanning (as your reverse primer appears to be) to ensure you selectively amplify from cDNA and not parental genomic DNA (present potentially in small amounts in your purified RNA used for RT, unless DNase digested before carrying out RT)
• Thus, a forward primer internal to an exon but a reverse primer exon spanning should only lead to amplification from cDNA - in other words will be specific -  even if you didn't DNAse treat your purified RNA before performing RT

What are amplicons size?  For SYBR green assay you need short fragments. No more than 70-80 bp.

Thanks Hall for a well-elaborated answer. 

Product length is 172 bp. Does it happen to be something my experiment can't handle? 

In short yes. 70 to 150bp ideal but < 200bp is still ok. Much above that and PCR efficiency falls below 85% which can confuse calculations of relative gene expression levels. So yes that is on the 'high side' of fine

Make a positive control on your reference genome and go ahead

I tried modifying my primer location manually and now the product is going to be 158 bp. Thanks a million for the advice!

What shall I do when NCBI says this " Your PCR template is highly similar to the following sequence(s) from the search database. To increase the chance of finding specific primers, please review the list below and select all sequences (within the given sequence ranges) that are intended or allowed targets"

Hi again. 

• If same species and complete match you will get non specific priming so design alternative primers 
• If same species but a mismatch that falls within  the last 5bp of primer sequence redesign primers
• If identical but with mismatch outside last 5pb ignore but make sure during PCR You use an annealing temp Tm C to Tm-2C
• Even if 100% identical but same gene in a different species obviously ignore 

Hi,  Thanks for replying to the query. Is it true that oligoanalyzer (IDT) and premier biosoft do not follow each other for calculating delta G? I was quite happy with my delta G using premier bisoft and I don't know why I thought of verifying it with IDT. Now they are appearing more negative. Like biosoft showed SD -3 and with IDT I got -6? Which tool is more reliable?

It's really what Jack Gallup mentioned: all of these algorithms are subtly different and so yield different values and as Jack also said are highly theoretical: to reiterate my previous point unless you have 3 consecutive G/C residues at the end of each primer or complementary GC residues within a primer resulting potentially in a hair pin I would simply ignore. What is more with DMSO and higher denaturing temp I wouldn't worry at all

My final piece of advice would be to put your primers through Northwest oligo calculator to investigate secondary structure: rather than confusing delta G values they simply have hair pins or no hair pins. 

See my primer design doc in previous answer 

Thank you so much. Yeah, I read your document. It is a great piece. You are amazing!

Kindly add your answers to this post of mine.

https://www.researchgate.net/post/I_am_looking_for_stable_reference_genes_for_RT-qPCR_If_anyone_can_suggest?_tpcectx=subscription_feed&_ntpl%5BactivityCombination%5D=person_add_comment_question&_ntpl%5BactivityId%5D=870679578808320&_ntpl%5BactivityTimestamp%5D=1502027387&_ntpl%5BactorId%5D=12448639&_ntpl%5BobjectId%5D=59871e7bf7b67eb21c4fc2b5&_ntpl%5Bs%5D=2e49147c0e5fc750f65ab937927a9eea65c08986&_ntpl%5BtargetId%5D=5986f19e404854a7766999e8#59871e7bf7b67eb21c4fc2b5

@Laurence. I know the disscussion is old, but I have a question. When you said:

• "If same species but a mismatch that falls within the last 5bp of primer sequence redesign primers
• If identical but with mismatch outside last 5pb ignore but make sure during PCR You use an annealing temp Tm C to Tm-2C"

Sorry if I'm not right, but shouldn't it be the other way around?

I read your doc and I understood that last 5 bases give you specificity.

If there is NO mismatch within in the last 5 bases (ie, the mismatch is outside las 5 pb), shouldn't you redesign your primer? Since it will be "specific" for both sequences.

Alternatevly, if mismatch is within the last 5 bases you may be able to avoid non specific products if you use Tm to Tm-2 for annealing?

In case I'm wrong, could you please explain me?

Thanks in advance for your response.

Sulail Fatima, the showed values are great. The recommended minimum ΔG for hairpin is - 3 kcal/mol and for self and hetero dimer is - 6 kcal/mol. These values are proper for qPCR, for common PCR you can be less rigorous. The ΔG hairpin for primer acta2 is a little bit lower than 3 kcal/mol (- 3.2 kcal/mol) but this is not a problem. When i design my primers i use this parameters and it is very difficult not to work out. Let me know if the primers work out. The more ΔG value is negative the greater the probability of these inconveniences (hairpin, self and hetero dimer) occur.

Agree with@ Laurence Stuart Dawkins-Hall.

Kind regards