Have you used the same template for your primers and the published ones? I think it can caused by the contamination of the template only. If your primers are bad, than you must get signals in the "water and primers only" control, or never get any signal.

Hi Gizella, yes I used the same template for both. Contamination is indeed the easy answer but it just doesn't make sense since all replicates for the No RT control of my designed primers gave a signal similar to the reaction tubes, whereas for the primers from literature, none of the No RT control gave signal.  During my preparation, I cannot think of a systemic mistake I did that would allow this trend.  In any case, thank you very much.  

I often come across published primers that are not specific to what they should be (or do not bind on ANY species... came across that 2 weeks ago!). In general we've stopped taking primers from papers (sometimes you come across some good, but we've decided in most cases it's easier and less stress (and money) to design new one by ourselfes.

I use PerlPrimer (a programm that ist open source), get me my sequences from NCBI or ensembl, and then I blast the primers wich I get from Perlprimer with NCBI primer blast or the blat tool (? I believe the name was something like this...) on ensembl, and Beacon Designer Free http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1 to get data about self and cross dimers.

You can of course try and blast the human/mouse/rat/whatever primers you've found and hope they will bind on porcine too, but don't put your hopes too high. If the primers are very specific, it won't work, and if they are not, there is always danger of crosscontamination...

One last thing: I hope you ran a gradient PCR to optimize your primers? If the annealing temperature wasn't optimized, it may be no wonder that the peaks of NTCs and samples where the same...? Maybe the primers just did'nt bind to your templates because of the wrong temperature? (Oh and I would also do a gradient for paper-primers... every cycler is a little different!)

Good Luck with your PCRs!

The product in your "no RT" control is from the DNA, not RNA, even if you added a DNase treatment during or after RNA extraction. Have you done a melt curve on your samples? If the primers cross an intron the product in your "no RT" control should have a different melt curve profile than the rest of your samples. 

Dear Christine,  here are some suggestions:

1.) "The problem is when I ran my primers, I got a signal in my "no reverse transcriptase" controls that have the same peak at the melting curve as my expected amplicon."

Run the product from the NTC and from a positive control well on a high% agarose gel and check the size. If the size is below 50bp, most likely you see primer dimers (which are more or less normal in the NTC, but also appear if any of your samples have not template expressed to be amplified.  If the products are between 80 and 150 bp (as predicted from the target sequence), then you definitely have a contamination in the NTC. The contamination will come either from post-PCR products of the same gene, or in case you cloned the same sequence in plasmid and worked with it on the same bench using the same pipettes, racks etc etc.  This is a real nasty problem. Usually the only solution is to go to another lab (that has never "seen" an amplified PCR product or cloned fragment of your target gene). Contamination by genomic DNA, in contrast, is very unlikely.

2.) " ... it doesn't seem to be a primer dimer because the peak is the same as for my reaction tubes."  If your reaction tubes have no amplifiiable target (i.e. zero expression of the target gene), you will get the same type of primer dimers as in the NTC.

One thing that always makes problems are residual random- or oligo-dT primers from the reverse transcription. They are easily carried over toegther with the cDNA into the qRT-PCR tubes. They can make funny amplification curves with an early rise in the signal (at around 10 CTs) and a second rise (at the expected CT value, see the left curve in the picture below).  The funny thing is, that the first rise of the curve even happens WITHOUT any qRT-PCR primers, since it only uses the carried-over reverse transcription primers. If you have something like this, dilute the cDNA 1:5 instead of using the indeluted from the reverse transcription. Or purify the cDNA from all other reverse transcription components, using QIAgen spin columns.

3.) For qRT-PCR primer design I usually use an on-line program from GeneScript. Although it only has inbuildt mismatch tables for human, mouse, rat and "others", I guess if you use human for your pig-genes, it should work.

(https://www.genscript.com/ssl-bin/app/primer)

Thank you Michaela for the input.  That is also why I decided to just design my primers as well.  I did run a gradient. Thank you for the well wishes, I hope I figure this one out soon!

Hi Lisa, I've been coming across that advice about designing primers that cross an intron, but I don't know how to properly do that.  You see I'm using this "consensus sequences" I got from a pig genomic database (that I mentioned above in my question), and I believe what they have there are the coding regions only.  If I include an intron, will it still be possible to keep amplicon sizes between 80 to 200 (as advised by my BioRad SYBR Green Kit)?  I was trying to look for a website that describes a method in a step by step manner but couldn't find any.  I have no background on biotechnology and all of this is new.  

Hi Michael, thank you so much for the detailed suggestions.  I did run my PCR products on an agarose gel and they are of the same size of the expected amplicon.  I have attached a picture of the two gels.  The first one is when I did using only the primers I designed wherein everything had a peak in the no RTC control.  It was my first time to do PCR that time and so I don't think it was due to any contamination of post-PCR products.  The second one is when I just used the ACTB I newly designed (this is a different ACTB primer design from the previous one), along with HPRT1 (Nygard 2007) and SADH (Nygard 2007).  Again there is a signal for my ACTB No RT controls however for both HPRT1 and SADH there are no signals.  The lanes with "?" on top were reaction tubes that I think had primer dimer problems because they had two peaks so I checked them.

Thank you so much for all your help! I really appreciate it.

Hello Christina,  the signal in the ACTB / no Rev Transc control is indeed a problem. To rule out that it comes from residual genomic DNA (as Lisa suggested) I would run a single reaction with pure genomic DNA as template only (5 ng). If this ampllifies the same product size, it is quite obvious and you must find better primers that span intron/exon boundaries. I had a look at the human homologue, and it shows 5 exons. I guess that you can take the location of the exon/intron boundaries from the human gene, and have a good chance that it is the same in pig. If you use the qRT-PCR primer design program from GeneScript (see above) you can define exon-intron boundaries in the sequence (in FASTA or text format) simply by putting in a colon between the two base paires (like   ......gcaatag:atcagcag....).

The issue with the melting curve double peak might be a minor problem. In the gel, a very weak secondary product is sometime visible (in lanes marked with an ?).  Sometimes  the qRT_PCR software is over-sensitive, and gives an error flag for double melting peak already at an too early stage. below are two picture of melting curves, both had an error-flag from the software. Whereas the first one has really a strong secondary product, in the second one the unspecific product was weak (only about 10% of the specific one).

So not always can one 100% trust hwo the program interprets the melting curves.

good luck with the further experiments,

Michael

Hi Christine, I just want to mention again (because you asked how to do), that the program I use (PerlPrimer, its free, and runs on Windows, Linux and iOS) helps you with the intron/exon problem... you just need to put in the genomic and the RNA sequence, adjust the settings as you want them, and the programm will calculate the introns and exons and will suggest primer that follow all the rules you set before (Like amplicon size, exon spanning by x bases, Ta,...)

Good Luck again, you will solve the problem!

Thank you again, Michael, for the detailed response and analysis.  I read through more about primer design spanning exon-exon junctions or including introns and am currently redesigning my primers.  My problem is that some of the genes I would like to get primers for, the exon/exon junction or intron inclusion doesn't work. But thanks so much for the advice, and also that information about melting curves.

Thank you Michaela, I'm trying to stick to Primer3 or NCBI's Pick Primers because that's what my BioRad kit recommended.  I don't have a full grasp of these things so I don't want to modify too much in the settings in looking for primers.  Kinda pressed on time to get data ASAP. Thank you so much and all the best in your work too!