Oh, and I also menat to say that my Ct values are also very high (28-32)

Hi, Lindsey.

Do you have a picture of your electrophoresis of cases when you saw unwanted products? Might help us.

As this is a RNA-based experiment, did you check the RNA integrity?

What's you product's Tm according to your experiments?

According to your primer sequences, I don't see any 'dangerous' nor even significant dimerization predisposition of the pair. What's with the negative control (no sample added to the PCR mix), do you see amplification there? Performing electrophoresis of the negative control along with 1-2 samples will help to decide whether there is the amplifiable dimerization.

Ta of 61 is rather high, indeed.

I'd suggest trying touch-down PCR. Reducing the magnesium ion concentration may also help (gradually down to 1 mM, for example), but this will also reduce the product yield. Finally, redisign primers, if the present primers are unreliable in the end. This may also be the cheapest way in terms of both time and money.

Hi Peter,

Thanks for your excellent response. I haven't been able to do electrophoresis as the lonza cassettes we use have run out, and for some bizarre reason normal gels are coming up blank (but ladders are fine). This is also not helping me problem solve. I always nanodrop my RNA before I use it and 260/280 values have been ok. I am always seeing products at around 74-75C in the melt curves for the no template control, hence why I am thinking the problem I am having is with primer dimers. I would have to get a new kit without a mastermix to vary the magnesium wouldn't i? I haven't read much about touchdown so I'll check to see if our machine does it. I have no idea what the tm of my product is (how can I find this out?), but the length is 124 bp.

Thanks again.

Dear Lindsey,

Blank gels is my 'favourite' thing about working with RNA. For me it has always meant RNA degradation of samples and oxidative stress of mine :)

And in this sense, 260/280 has almost nothing to do with the RNA integrity: it merely reflects extraction purity and even in this application it is not as accurate as we'd want it to be. In this regards, please see related topic on RG:

https://www.researchgate.net/post/Comparison_of_Nano_drop_and_RNA_integrity_number_RIN_data

and

http://www.umich.edu/~caparray/products/sample_prep/fleige-pfaffl-rin-2006.pdf (good references there)

You should better run an ordinary (even non-denaturing) gel (1.5-2%) to look at your RNA.

Seeing products in the negative control probably suggests contamination of reagents or labware, as Tm is high enough to represent a normal product, not dimers. You can calculate your product's Tm (at least relatively to possible dimers, though I consider the amplifiable dimers risk very low) by entering its sequence into: http://www.basic.northwestern.edu/biotools/OligoCalc.html

Please also consider that your forward primer has 4 Ss (C/Gs) in a row, thus having a powerful non-specific clamp.

And yes, further optimization may require having separate reagents, not the master mix. However, you need to be sure that what you are dealing with is not an ordinary contamination.

Do you use hot-start PCR? As you are dealing with a complex task of working with RNA extraction, reverse transcription and PCR, each step may be a problem, so we all probably need more details on your steps.

Thanks all.

Peter, your comment has given me something I hadn't considered. What has been confusing about the gels is that when I use lonza cassettes I see bands, but the normal gels come up blank (despite the ladder being fine). I even got trained in running gels again to make sure I wasn't doing anything stupid!

I will run a gel, but as I'm not familiar with this can you explain to me what I would be wanting to see in degraded and intact RNA? Would you suggest doing a normal agarose gel?

I know this may sound ridiculous but I don't know the sequence of my product. I have been wanting to check the tm and have been trying to get the sequence to do so but primer blast only gives the length. Yes, I think I am using hot start pcr . I use the qiagen mini kit to extract the rna and do dnase digestion, use the omniscript rt kit for cdna conversion. In the qPCR I do initial activation at 95C for 15 min, initial denaturation at 94C for 15s, annealing at 61C (and the other temps I've used) for 30-35 cycles, extension at 72C for 5 seconds and then a dissociation curve.

Thanks both for your help

I also meant to add that whilst I am focusing on just one gene currently, I am actually trying to validate quite a large number of primers relating to five different lineages (so am using 5 different sets of RNA). Although I haven't looked at the other primers yet in as much detail, I am seeing the same problem with all of them so far (they all have a peak around 74-78C). That I've been attributing to primer dimers. Peter, you suggest that this may actually be too high to be so - what is typical for primer dimers then?

The dimer seems to be coming from annealing of the terminal 2 bases between your primers -- Forward AG.... with Reverse ....CT. See if there's any way you can avoid that terminal complementarity.

Lindsey, I'm working with animal RNA so I rely upon 28S/18S rRNA bands electophoretic analysis. But the basis is the same for our cases:

http://www.lifetechnologies.com/ru/ru/home/references/ambion-tech-support/rna-isolation/tech-notes/is-your-rna-intact.html (see Figure 1);

with the difference that you will see and need to analyze 23S and 16S rRNA:

(please see p.5 - RNA integrity section) http://www.gene-quantification.de/aranda-rna-quantification-2009.pdf

For the routine RNA integrity check you can run an ordinary non-denaturing 1.5-2% agarose gel. Denaturing gel would make the bands look sharper, but this is not the most important thing, while preparing such a gel is far more complicated. You can also run a sample composed of 60% formamide and 40% RNA in a non-denaturing 1.5-2% agarose gel - the bands get sharp.

I would respectfully disagree with John's idea: AG/CT pairing gives one of highly unstable possible dimers with the length of 21 nt and a product of the same length (only forward primer is to be elongated). This dimer doesn't look any close to give a product melting at 74-75 DC (calculated salt-adjusted Tm is 61 DC). Please see the heterodimerization analysis file attached.

I work mostly with semi-quantitative RT-PCR, so I cannot provide a statistics on primer dimers melting temperatures (this is why I suggest running a gel - dimers are very easy to find there) and I haven't seen dimers for a whi-i-i-ile. According to calculations, for example, a Tm of 75 DC is characteristic of a product of 30 nt (GC 57%), which means that a primer dimer must have a 6 nt complementarity stretch, which would have a Tm of approximately 25 DC. Thus, if you are using hot start, this should not be a concern. Especially given the fact that you don't actually have any dimers with such a high complementarity.

Considering that you have the same problem with all of your primers, I'd suggest making sure you are not really dealing with contamination. Wash the laminar cabinet and all pipettes according to the manuals, change all the plasticware, use new reagents and fresh primers prepared from frozen stock. And be sure that you do actually have RNA.

I've forgotten to tell you about touch-down last time. It's a very simple procedure when you assign several sets of PCR cycles with decreasing Ta. For example, 5 cycles of Ta of 60 DC, 5 cycles of Ta of 59 DC, 5 cycles of Ta of 58 DC, and so on and as you like it. Sometimes it really resolves non-specific amplification.

Hi guys,

I have had some success. I have used a fresh kit and now at least two of my primers (including the one I was struggling with that prompted me to write this post) are now working fine! Thanks for everyone's help.

Following optimizing the annealing temp, [primers] and quantity of RNA to be used (the new kit is a one step process), I will need to check the reaction efficiency. I am sure this is a silly question - but does the reaction efficiency need to be checked again if another kit is used to the one used to test efficiency? I ask as I am about to go on placement elsewhere and do not know what kit they use.