Laurence Stuart Dawkins-Hall

Many thanks for your quick and helpful answer.

• We already tested our samples with other probes from Bio-Rad (ready-to-use PrimePCR assay). For that we received a quite good curve.
• Unfortunately, we only have RNA samples from blood.
• We use CFX Connect as cycler from Bio-Rad. For our measurements, we chosed the 'PrimePCR' button and performed our run with these background settings (cycler run condition was modified to the used master mix).
• In our group, we are the first one, who want to implement probe assays with that cycler. Maybe, the fluorophore calibration is not correct?

Laurence Stuart Dawkins-Hall

Many thanks for your answer.

• The ready to use probes from Bio-Rad that yielded good curves detected houskeeping genes.
• What should we change at our probes when we want to reorder them? Completely redesign? We designed them according to the eurofins manual (e.g. Tm >8-10 °C higher than Ta of the product,...) and they seem to be specific in assays where we used plasmids as positive controls.
• Maybe the quencher at the 3' end is not the best choice...but we ordered BHQ1 for FAM and Hex probes?
• The ready to use probes from Bio-Rad as well as the probes from Eurofins come in tubes.

Laurence Stuart Dawkins-Hall

• What I forgot to say...due to evaporation we used caps instead of foils to cover the wells

One issue I see is that the RFU values are very low. You should be seeing much higher signal. Make sure that your reagents are fresh and have not been frozen & thawed more than twice total.

Laurence Stuart Dawkins-Hall

Many thanks for your answer and the literature

• Until now, our houskeeper probes/primers are from Bio-Rad

Katie A S Burnette

Many thanks for your answer.

• We used freshly reagents and stored the probes/primers/master mix at -4 °C in the fridge.

Catarina Figueiredo Mota

Many thanks for your answer.

• We used the CFC connect as cycler from Bio-Rad and the GoTaq Probe qPCR Master Mix from Promega because in our tests, the master mix from Bio-Rad did not work well
• For the housekeeping genes we gained Cq values between 20-25 and the curves were fine.
• We used FAM and HEX as fluorophores and BHQ1 as quencher. For the Bio-Rad one, we are not sure whether they used BHQ1or not. But we are waiting for the answer from the Bio-Rad service.
• The only probes that we used from Bio-Rad are for housekeeping genes and OR51E2.
• The Bio-Rad probes did not show such strange curves that „goes up and down“.
• We think that our cycler is compatible to that plates, caps and fluorophores due to the settings. We still wait for answers from Bio-Rad.

Catarina Figueiredo Mota

Many thanks for your quick answer.

• Yes, that's true. But from Bio-Rad, we have only probes for housekeeping genes in our assay and for OR51E2 with a moderate curve and Cq values around 33.
• We did duplex-qPCR with FAM and HEX with our cycler
• That's a good idea. We will check the band on a gel.
• We can change the cycles to analyze from 1-40 to 3-40.
• The Bio-Rad service center assumed, that we have problems with the lid (pressure or heating) because when we used sealing foils, they didn't close the wells well (they didn't melt around the wells)...even with new foils...therefore, we used caps instead of sealing foils. Due to the high specificity and sensitivity of probe assays, the Bio-Rad service center assumed that we are only able to detect such strange curves in probe assays instead of SYBR assays...Can anyone of you imagine that phenomenon?

Catarina Figueiredo Mota

Many thanks for your comments.

• Using the sealing foils MSB1001 of Bio-Rad, we detected volume loss
• I mean, that we can cut the first three cycles away and therefore the curves seem to be "better". The curves start at cycle 3 until the end. I don't mean baseline correction
• Yes the plot I showed you was before any corrections. Yes all samples were measured in duplex conditions and only probes from Eurofins were used in that plot.
• In the attachment you will find the modified plot and more graphs of different measurements
• Iwe could detect our 10 targets with SYBR, but we only received low CQ values but a better curve (see the attachment)
• Yes we try the probes in single conditions as well. You will find one measurement with that condition.

Catarina Figueiredo Mota

Many thanks for your answer.

• I uploaded a file with colored graphs of the measurement with different probe/primer concentrations as well as of the measurement of 10 genes of four patients. The gel belongs to the last one.
• We also manually set the baseline to 5-15 and the curves look great (see the attachment). So we have to improve our settings.
• On our gel, we are able to detect the assumed amplicon sizes of the 10 different genes.

Catarina Figueiredo Mota

Thanks a lot for your help.

We will further validate our assay.

• Today we implemented a probe assay with additional 3 % DMSO final conc. to improve the annealing of the primer/probe to the template and to exclude secondary structures
• What we observed was that until cycle 20 the baseline was flat and only the two housekeping genes were amplified. Later we had a further look onto our cycler and what we obserbed was that we had such a "dropping" baseline. And only at this time we observed a small increase of the baseline at cycle 4 (see attachment, black arrow). So at the beginning the baseline was really flat...
• We will try your suggestions.

Hi, Catarina Figueiredo Mota

I just saw your conversation and checked the literature and find out what probably happened. I already have seen it happening and, in the case I saw personally, it was in a ABI7500. We got the same strange curves and discussing with thermo support we didn't get a clear answer too.

Probes there were compatible to the equipment and they did experiments to validate their results without success.

Here you will be able to read the important data about this problem and solve it, at least, that's what I hope.

Link: https://www.mlo-online.com/home/article/13008268/interpretation-of-qpcr-curve-shapes

https://www.sigmaaldrich.com/technical-documents/articles/biology/troubleshooting.html (another nice reading about it).

Although I guess you're not having evaporation on your samples during reaction, it is a possibility as you can see here: http://www.aun.edu.eg/molecular_biology/Procedure%20of%20PCR%20level%202%20(5.7-4-2016)/troubleshooting%20of%20RT%20PCR.pdf

Another info saying and showing the same results can be seen in the following link:

https://www.idtdna.com/pages/education/videos/qpcr-pcr/videos/default-source/webinar-videos/qpcr/troubleshooting-qpcr---what-are-my-amplification-curves-telling-me

One last thing ... Looking into your gel, we can see two and three different bands in different lanes. So, your reaction is not specific by one of three major possibilities:

1) Maybe you need to standardize your reaction yet (changing annealing temperature);

2) Your primers are getting different splice variants. Please, check them again in order to confirm their specificity. Ok?

3) Too much template is giving you a baseline that influences the graphic results and, at the same time, different amplification efficiences, giving more bands than it should.

Hope it helps and I will check what was my lab partner's conclusion of what happened in our experiment here.

Adriano Costa de Alcantara

Many thanks for your answer and the respective links. I have read them.

Due to our implemeted dilution series of template cDNA (2.5 ng to 20 ng) in which we received for all amounts of template the same strange curves, we think that we have a software issue. We are limited in template amount because it derived from patient blood samples and therefore we cannot use more than 20 ng.

We will use an other cycler from our partner group and will test our probe assay there.

Maybe we can solve our problem. I hope.

To your question:

This would be great.

Greetings

A agree too Adrianos comments.

Hi, Tamara.

I would team with Catarina and ask you to include what happened here because other people who get in the same type of problem can get some light into it. Ok?

This is one of the things I think ResearchGate can include as a request in order to make it even more helpful for the scientific community.

Resolution is the major achievement and data for other people is the only way to help them all (even ourselves). Ok?

All the best for you and all the others in this post.

Catarina Figueiredo Mota

Sure, I will tell you the results, but until now we did not solve our baseline problems! Therefore, I did not write anything here!

We tested an other cycler but with that we received also very strange baseline curves.

Therefore, we contacted Eurofins that synthesized our ologonucleotides. We assumed that something went wrong with the synthesis of them.

We are also in contact with Bio-Rad.

We are still waiting of answers of them.

One company assumed, that our oligos are too long (25-35 bp). But we designed them with the "Eurofins Design Tool" and Eurofins confirmed, that their tool are a good choice.

Greetings

Tamara

Hi, Tamara.

Although you said you did a dilution of your cDNA, we should not use cDNA as sample for measurements of amount of starting material. How much did you use for reverse transcription? 1ug or 500ng?

Secondly, 2.5 to 20ng is to close as it will only vary 10 times, which means 3 cycles. So, If it is too much starting material, it will keep on being close to the limit of higher detection, giving these strange results. Ok?

I think it is worth trying to perform a higher dilution to check if it changes. I would do at least 20x and 100x in order to get rid of any doubts. Ok?

Hope it helps.

Catarina Figueiredo Mota

Many thanks for your answer.

We are in close contact with Bio-Rad and Eurofins.

• Today, we had a call with Eurofins. They confirmed our findings that our baseline was very strange and that the housekeeping genes from Bio-Rad were better. They will further analyze our probes and then we will hopefully get new one from them. Maybe there are "free dyes" or "not working quencher" in our ordered probes.
• Moreover, our cycler will undergo maintenance again due to the "lid problems". We will also receive a new cycler from them as testing device.
• The other cycler was from Applied Biosystems (Quantstudio4).
• The Bio-Rad probes (housekeeping genes) showed a flat baseline. Yes we tested them all in singleplex, but we did not select each target independently to determine the automatic baseline.
• Yes, the same primers performed well in a SYBR assay.
• We made RT-PCR and we received for every amplicon one single band.
• No, we did not see any differences between the shorter and longer probes.

Adriano Costa de Alcantara

We were very limited of our starting material. It's from patients and differed between 20-100 ng/µl in 150 µl total volume. The samples were stored as neutrophils in RNAprotect for more than two years in our -80 °C freezer and until now nobody had implement assays with them. We had also freshly isolated RNA from blood bags with 176 ng/µl RNA.

Ok. Thanks.

Hi, Tamara.

Thanks for your answer ...

So, two things you may do to check what I told you ... amount of total cDNA in the qRT-PCR reaction. Because you are short on sample, you could use a pooled sample to check it while you're waiting the response from the companies. Ok?

Secondly, how much RNA for all your samples have you used to perform the reverse transcrption? 100ng, 250ng, 500ng, 1ug???

Waiting for your answer in order to help.

Adriano Costa de Alcantara Catarina Figueiredo Mota

Many thanks for your answers.

We had samples from blood bags for testing our probe assay. From that we had enough but we want that our assay work as well as with low amounts of RNA of the patients.

We had used a high capacity kit from Applied Biosystems and for that we used around 500 ng RNA per reaction, in average. Therefore, we had more reactions to do per RNA of each sample.

Eurofins will re-synthesize our probes and we will receive a new device from Bio-Rad for testing our probe assay. We hope that we can do that in the next two weeks. Then I will report the results, here.

Hi, Tamara Krämer

Thank you for your response and I am wishing you all the best with the new primer/probe synthesis and experiments.

I'll be waiting your results to see what happens.

All the best.

Adriano Costa de Alcantara

Many thanks.

We are looking forward to the results.

Greetings

Hi, Tamara Krämer

I found out my partner has posted here the problem I told you. So, Unfortunately, It did not reach a final understanding of what has happened ... But there was a comment from two researchers (Jochen Wilhelm and Barry Atkinson) which seems to corroborate with the possibilities you have been told. You may check it at the following link:

https://www.researchgate.net/post/What_causes_the_wiered_q-PCR_curve_with_Cy5_on_7500_system#view=5b9645274f3a3e88ab01b491

Waiting your future comments of how you solved it!

All the best.

Adriano Costa de Alcantara

Many thanks for your answer and the link.

We are looking forward to solve our problem :-)

Hello everyone,

we received the re-synthesize of our probes and we also received a new device from Bio-Rad for testing our probe assay.

With our standard cycler program (95 °C, 2 min; 95 °C, 15 sec; 60 °C, 60 sec) that fits to our used master mix from Promega, we received the same disappointing values and baseline with the new cycler and the new probes.

Then we changed some settings and used another cycler protocol and we received a very nice baseline (see attachment).

Unfortunately, we were not able to repeat this assay with the same results until now.

Have someone an explanation for that?

Many Greetings

Tamara

Hi, Tamara Krämer

I am sorry to hear you're still having problems with your qRT-PCR.

Let me ask you a few things:

1) After performing your cDNA synthesis, you're diluting your cDNA and using12.5ng per qRT-PCR reaction. Is that right?

2) Are you doing your probe signal detection on extension step? It should be performed on annealing! If so, test it doing the plate read, differently of what you did on your documented protocol to us, changing it to the 50ºC step. Ok?

3) What code of your GoTaq mastermix is the one you're using? Maybe you have a one-step kit instead of a qPCR one and it is performing a second 10 minutes cDNA synthesis, which may be giving you problems.

So, I'll be waiting for your answers in order to try to figure out what is happening. Ok?

Bye.

Adriano Costa de Alcantara

Many thanks for your answer and advice.

Catarina Figueiredo Mota

Many thanks for your answer and advice.

Greetings

Catarina Figueiredo Mota

Many thanks for your answer.

We can manually change our baseline to each well.

At the attachment, you find the probe assay with the 5-step-protocol that I already showed you and the new measurement from today with the same automated settings. For that measurement we did not receive a flat baseline.

At the attachment, you will also find a dilution series of one cDNA and one target in triplicates.

Catarina Figueiredo Mota

No problem. Until now you helped me a lot.

I agree with you, that the plots look almost equal. When you look into the plot that is converted into pdf then it seems that the first one showed a more flat baseline than the second one. We repeated this assay very often and we were not able to detect the same targets in triplicates with the same template...

We checked the wells for evaporation and it seemed that the liquid level was not altered at the end of the measurement. We also checked every well for air bubbles before starting the measurement.

We will further establish our assay and then I will Show you our results...

Catarina Figueiredo Mota

In the attachment you can find the plots converted to pdf and maybe my explanation is easier to understand.

Hi, Tamara Krämer

It is really strange but hard for us If we can"t take a look at the data as a file ... If you don't mind and wish, send me one archive from your results (as a cfx file) and i can try to figure out something and check at least If I can detect any problem on it ... What do you think?

If you wish, you can send it here or ask me for my e-mail by messaging me through researchgate webpage. Ok?

All the best.

Adriano Costa de Alcantara

Many thanks for your help.

Yes, I understood.

That's very nice of you. I only want your help when you have enough time. I have to solve the issue for my own but I am very happy when someone can help us to get rid of that strange issue.

I can send both measurements to you that we did with the same cycler settings and where you can see that both plots did not show the same "flat" baseline.

I can send it as a pdf, it's that ok? We have only the choice between pdf, bio-rad file or excel files....Which you prefere?

Thanks a lot for your help and many greetings.

Hi, Tamara Krämer

I do have the CFX program so, If it is not a problem for you and your team (advisor, PI, or chief), send me a CFX file. This way, I will be able to check a few things by myself and, If something is wrong with your analysis settings, I may tell you. If not, then, there is something wrong in the reaction or primers/probes ...

Check with your personnel, mainly your bosses, if they are ok to let you send me a file with your data. They may be worried about sending data out. Ok?

All the best.

Adriano Costa de Alcantara

I will ask my chief. Thanks a lot for your help.

Catarina Figueiredo Mota

Many thanks for your answer.

• Yes we did so.
• We checked every primer & probe combination using the respective plasmid when we had it in-house.
• We will continue with our tests. You have right!

Hi,

we tested the new test device from Bio-Rad with sealing foils instead of caps and we received a nice flat baseline. We were able to replicate such baseline results and we decided to use that device for our patient samples.

With our Bio-Rad device, it was not possible to use sealing foils. Here, the sealing foils did not close the wells correctly and we had a lot of evaporation. Therefore, we used the caps.

Nevertheless, thank you all for your help.

Adriano Costa de Alcantara ,for the first time we will measured our patient samples with the new test device and therefore, I will not send to you our Bio-Rad files. I hope you understand my decision.

Greetings, Tamara

Hi, @tamara.

Great news!!!! So, in your case it seems to have been evaporation issue. Easy to solve, now that you know what is happening. Congrats!

About your decision, of course I understand and that's why i told you to ask your PI before you sent it too.

All the best to you, your team and project!

Adriano Costa de Alcantara

Many thanks for all your help.

HI, Tamara Krämer

You're more than welcome!!!!

Good luck!

Thanks a lot!!!!

hi Tamara,

could you please comment on your graph why dont you have plateau phase . i have the same issue with some primer.

Hiresh Ayoubyan

With new probes and sealing foil instead of caps we received a fine baseline. For our HKG we get a plateau but not for our targets of interest. Here I would say, that we have not so well expressed genes and we only received signals after cycle 25. This signal did not reach a plateau phase until cycle 40.