Here is the melt curve..

Smears in a gel are often due to high salt concentration.

Use a program such as Primer 3 to design your primers optimally. Reduces risk of self-complementarity.

Bear in mind that your primers are more likely to start amplifying each other if a DNA target is not available through either low expression or poorly designed primers.

Keep amplicon length short, up to 150bp.

Use hot start polymerase.

Keep samples on ice.

I used ready to use kit and can't adjust the salt concentration. I used 100nM final concentration for the primers. If the expression is low, but should be able to detect by real time RT-PCR right? the amplicon size is around 160bp. I did use hot start polymerase.

Dear Shin Wei Tie

I recommend you to reduce primer concentration (forward primer .7 microliter and reverse primer .4 micriliter, of course, for 10 pico mole concentration of primer, I already had the same experience. I did touchdown, and I got good results, please try to do these two comment (primer concentration and touchdown).

Reyhaneh, what do you mean by 0.7ul & 0.4ul forward and reverse primer for 10 picomole concentration? Does it mean 10picomole final reaction concentration in one tube?

Dear Shin We Tie

I mean that It is better for you to perform PCR with unequal F & R primers concentration, Primer stock concentration for doing PCR is usually 10 µM , So, take 0.7ul & 0.4ul from these stocks)

Example: If you use 1 µl of this primer stock (10 µM solution) in a 20 µl PCR mix, you are using 0.4 µM of primer in the final mix (final reaction concentration in one tube).

let me know if you have any more questions.

Dear Reyhaneh,

I have tried the concentration that you suggested and touchdown, but the peak still not much different. I use final concentration of 350nM Forward Primer and 200nM Reverse Primer.

Since it sounds like you are doing everything to minimise primer dimer formation, we need to establish whether we are actually looking at a primer dimer or not -If it is a primer dimer then you will also see a peak in the water alone sample. This peak will be in line with the 'shoulder' that appears on the main peak you showed above. If you don't see that then we are not looking at primer dimer. The smear you see in your gel could just be the primers, not amplified?

It is possible for single PCR product to give two peaks (overlapping in your case). This happens when there are two very different regions in your product such as a stretch of AT-rich ( which will melt at low temperature) or a GC-rich region (which will melt at a higher temperature). Does your amplified region look like this?

As long as your water doesn't have this peak, and all of your samples have the same shape of peak, your quantitation will be OK.

Amanda, you mean only water with SYBR Green MasterMix and primers in the tube without the enzymes?

I mean water instead of cDNA. If primers are being amplified then this sample will have the highest level of dimers. The more cDNA you add, the less primer dimer will be present because of the competetion between your target gene and the primer. This is what I was trying to say before - yes you can measure small amounts of expression with real time PCR, but you need to have very good primers since poorly designed primers may prefer to amplify each other rather than search for your very small level of target gene in the sample.

Amanda,

I did get the primer dimer peak in the No Template Control tube. around 78 deg. Celcius.

Definitely a primer dimer interfering with your quantitation then. And since you have done all the things I would recommend to reduce this, without success, I would suggest designing different primers. I use Primer3, available free online, to design all my primers. Just be sure to either span an intron exon boundary with one of your primers or else put each primer in a different exon ( I do the latter) and change amplicon size to less than 150bp. I am using one step RTPCR sybr green kit (Qiagen), 40ng RNA, 300nM primer in 10ul, 57oC annealing temperature with all my primers designed this way and I don't see any primer dimer, even in no template control. If any of my primers are questionable in any way, I throw away and order different sequence- its much quicker than trying to fix.

Dear Shin We Tie

use final concentration of 140nM Forward Primer and 300 nM Reverse Primer, and start touchdown from 70 oC , (reduce temperature 0.5 oC during 10 cycle, and use 60 oC or 59 oC ( 15 seconds) as annealing temperature, (PCR 35 cycles).

please try it and let me know about result.

Dear Shin We Tie

in addition, it is better to dilute your RNA (two fold), of course first, do qRT-PCR without dilution (as I mentioned above), if you didn't get good result , go for diluting.

Dear Reyhaneh,

May I double confirm with you the touchdown is from 70 deg Celcius, and decrease 0.5 deg Celcius/cycle for total 10 cycles right?

Oh! excuse me! ( 1 deg Celcius/cycle ), I mean that try this procedure: start from 70-69 deg Celcius and continue to 10 cycles, then start your main PCR with 60-59 deg for 30-35 cycles. of course you can start from 70 oC and continue 20 cycles (decrease 0.5 deg Celcius/cycle ) and then start your main PCR with 60-59 deg for 20cycles.

If the results were slightly better, you can adjust temperature and number of cycles. you can reduce and even remove Primer dimer from reaction. I could do it, so can you.

Dear Reyhaneh,

Here is the details of the curve. Perhaps is a good news but I think there is a small peak beside the desired peak at 79.5C. The cycling protocol is as below:

Cycle 1: (1X)

Step 1: 42.0 °C for 20:00.

Cycle 2: (1X)

Step 1: 95.0 °C for 05:00.

Cycle 3: (20X)

Step 1: 95.0 °C for 00:10.

Step 2: 70.0 °C for 00:15.

Increase set point temperature after cycle 2 by -0.5 °C

Cycle 4: (20X)

Step 1: 95.0 °C for 00:10.

Step 2: 60.0 °C for 00:15.

Data collection and real-time analysis enabled.

Cycle 5: (1X)

Step 1: 95.0 °C for 01:00.

Cycle 6: (1X)

Step 1: 55.0 °C for 01:00.

Cycle 7: (61X)

Step 1: 65.0 °C-95.0 °C for 00:10.

Increase set point temperature after cycle 2 by 0.5 °C

Melt curve data collection and analysis enabled.

Your primer dimer looks more significant in this reaction. The primer peak is greater than before and the specific product peak is lower. A large amount of quantitation of sybr green will come from your primer dimer.

Dear Shin We Tie

Do you treat RNA with DNaes ? I think It is better to change all of components of real-time PCR and template and repeat PCR with reduction of 1 oC/cycle, let me again look at your results specially your agarose gel image. I think touchdown was incorrectly performed, because this procedure remove non-specific bands, but you got nonspecific ! (or primer dimer, of course I think this peak isn't primer dimer, because it is sharp and...), please also load this real-time PCR product into agarose gel and let me see it. thanks

@Reyhaneh The peak at 77.5oC is primer dimer because it is present in the no-template control as well as the samples. Unless there is a contamination of reagents that is amplfying with these primers. You're right though, touchdown PCR should have eliminated nonspecific priming.

I still think the issue here is that the target is too low to quantify, either because of poor cDNA synthesis or PCR, both of which may be fixed by using a different primer.

Dear Amanda O'Donnell

Please let me see his agarose gel image. I think this peak maybe due to primer dimer, contamination and..., However, , I would like to thank you for taking your time to read my answer.

Dear Reyhaneh,

I did not treat with DNase. The real time component are brand new. What do you mean by touchdown is incorrectly performed? Then may I know what is the correct touchdown? I will update you the gel image ASAP.

Dear Shin We Tie ,

I mean that touchdown remove nonspecific band, however, Please treat RNA, then repeat Touchdown PCR (reduce 1 oC/Cycle during 10 cycles and then 60 oC for 30 cycles), of course try with & without treatmeant and let me see both of them. (please repeat )

Dear Reyhaneh,

Here is the gel image. The first lane is the one done according to your advise. May I know what is wrong?

Sorry for the bad quality image..

Dear Shin We Tie ,

You're welcome, no problem.

I think that your PCR wasn’t successfully performed. Before carrying out the Real-Time PCR, it is better to do conventional PCR. You first need to make sure that whether annealing temperature of primer is correct or not! I always do this step before going for Real-time PCR with its RT-PCR kit. So carry out PCR with 2.5 microlitter cDNA (I mean that , do RT with 2microgram treated RNA, then gradient PCR for obtaining optimal annealing temperature for your primers ), After doing gradient conventional PCR , let me see your gel image.

Add Components of PCR (25 microliter final volume) as follows:

Buffer (10x): 2.5 microliter

dNTPs (10mM) : 0.5 microliter

MgCl2 (50mM) : 0.75 microliter

Forward Primer(10 pmol/µl): 0.8 microliter

Reverse Primer (10 pmol/µl): 0.5 microliter

Sterile Water (DNA-free water): 17.25 microliter

Taq polymerase (5U/ul): 0.2 microliter

cDNA: 2.5 microliter

Mix and microfuge all of them and then do PCR as follows;

Step 1: Initial denaturation: 94 oC for 4 min

Step 2: 35 PCR cycles as folloes:

• 94oC for 30 seconds

• 57-58-59-60 0C for 30 seconds

• 72 oC for 30 seconds

Step3: final extention: 72 oC for 4 min

Step 4: store at 4 oC for 5 seconds

After doing PCR load these product into 2% Agarose Gel and then let me see gel image.

Good luck

Dear Reyhaneh,

I had done conventional gradient PCR in January 2014 and this is the cycling condition and gel image.

94C, 3 min

94C, 45s

50-60C, 30s,

72C, 45s (40x)

72C, 10 min,

hold at 4C

I did TD-PCR also with the following cycling protocol and gel image:

94C, 3 min

32 cycles of:

94C, 45s

50C, 30s

72C, 45s

15 cycles of:

94C, 45s

50C, 30s

72C, 45s

72C, 5 min

hold at 4C

Refer to first four lanes including ladder.

I did 2 steps qPCR in March 2014, the cycling protocol is as below:

95C, 5 min

40 cycles of:

95C, 10s

60C, 30s (real time data collection)

95C, 1 min

55C, 1 min

61 cycles of melt curve analysis starting from 65C to 95C

This is the 2 steps TD-qPCR protocol:

95C, 5 min

15 cycles of:

95C, 10s

60C, 30s (-1C/cycle)

35cycles of :

95C, 10s

48C, 30s (real time data collection)

95C, 1 min

55C, 1 min

61 cycles of melt curve analysis starting from 65C to 95C

Dear Shin We Tie ,

did you perform my preceding protocol?(exactly step by step) if so and it wasn't good for you please treat RNA, then repeat Touchdown qPCR (reduce 1 oC/Cycle during 15 cycles and then 52 oC for 20 cycles), as follows:

95C, 3 min

15 cycles of:

95C, 20s

66C, 20s (-1C/cycle)

20 cycles of :

95C, 20s

52C, 20s (real time data collection)

let me see your images (gel & melting curve)

Dear Reyhaneh,

Thanks for the advise. May I know what do u mean by treat RNA? Why do you use 52C for annealing temperature?

Dear Shin We Tie ,

As mentioned earlier, to treating (Removal of genomic DNA from RNA): add DNase I enzyme and incubate it for 30 min at 37 oC, ( 1 microgram RNA+1microliter DNase I +1microliterBufer DNase I+ 0.5 microliter RNase inhibitor+add RNase free water up to 10 microliter) then add 1microliter EDTA(25mM) and incubate at 65oC for 10min.

You can perform with & without treating. please try to do these 2 ways.

Please find attached pdf file.

Dear Shin We Tie,

you can also add 1microliter 50mM EDTA. sorry I couldn't attach the file. Let me have your email address so that I can send it through the mail.

Dear Reyhaneh,

My email address will be [email protected]. Thanks for the suggestion.

What about this one? I am yet to run gel..

Dear Shin We Tie ,

Your primer dimer looks in this reaction. but results is better relative to earlier.

please let me see gel corresponding to this curve. Did you perform my protocol? or not yet? if not, please do my protocol , and again let me see gel image &melting curve.

Dear Reyhaneh,

I am following ur protocol.

Hello Shin Wei Rei,

I suspect the problem with your qPCR melt-curve profile to be a result of non-specific amplification than being a primer dimer problem (based on the first two gel pictures you posted along with the melt-curve profile). Did you blast check your primers for their specificity? Also, if you have a clone of your amplicon, you can try performing the melt-curve profile on that. It would clearly demonstrate the source of your problem.

Best luck.

I have designed a new primers and the melt peak of my positive control showed one peak at 80.5C and my another tissue sample has 2 peaks at 79C and 81C respectively. It is confirmed from the gel image that the second peak to the left is primer dimer for another tissue type and little amount of PD in positive control. The final forward and reverse primers concentration are 100nM and 300nM respectively. May I know if there is any solution to reduce this in my sample tissue? But why is the PD is obvious in my sample tissue but not positive control? (Positive control is HepG2 and sample tissue is THP-1 differentiated macrophage.) Is it may be due to low abundant transcript? 

Since your positive control sample shows a good single peak and band on the gel, your PCR conditions will be good and you shouldn't need to adjust anything there. As I said before, the appearance of PD is more likely if you have a low abundant target. It is possible that your tissue doesn't express this gene, or perhaps it expresses a variant or only expresses under certain conditions (I would highly recommend you do some searching on Pubmed, UCSC genome browser, Genecards etc to get some clues here). 

Be sure to check the quality of your tissue sample though. Do you have a housekeeper gene you can check expresses equally well in your two tissue samples.

You can run your RNA on a gel to check for quality too. (You can find how to do this and examples of how the gel should look on Google)

After doing all this, to finally answer your question, I would design several primers covering different regions of the gene and test them all. If it truly is not expressing, then it will be clear when you have a nice band in your positive control sample and not in your sample.

I still suspect the bands that you observe are not Primer Dimers but non-specific ampification. Consider that your primers are 25 bases each and so from end-to-end they make utmost 50 bp if they form PD, which is still a stretch. PD length would be less than the combined length of the individual primers. It is apparent from your gel picture that the bands are above 50bp. Probably your primers amplify isoforms. As Amanda O'Donnel mentioned earlier, you need to sift the literature for any isoforms known and design primers to be specific for your target. Also, you can sequence the non-specific band that is amplifying and that should help resolve your problem.