Hi Nailia,

here is some information missing to really answer your question. However, it seems that the PCR has not worked on your samples at all (the controls seem to have worked, but the signal comes up relative late). My first idea here is that you have a problem with your template - so my question is

1. What is your template?

2. How did you check the integrity of your template?

3. which enzyme mix/kit did you use for your real time PCR?

Basis for Determining the Temperature and Time of the Three PCR Steps • The three steps of PCR (Denaturation, Annealing, and Extension) are optimized based on the biochemical properties of DNA and the enzymatic activity of DNA polymerase. • Optimizing PCR temperatures and times ensures specificity, efficiency, and yield. Deviating from the optimal range can lead to incomplete denaturation, mispriming (primers bind to non-target sequences), non-specific amplification, enzyme degradation, or failed amplification. • The temperature and time for each step are determined by the following factors: 1. Denaturation (94-98°C, 20-30 seconds): ▪ Basis for Temperature and Time: » The hydrogen bonds between complementary DNA strands must be broken to generate single-stranded templates for primer binding. » The optimal temperature (94-98°C) is chosen based on the GC content of the DNA, as GC-rich sequences require higher temperatures due to stronger triple hydrogen bonds compared to AT pairs. » The time (20-30 sec) is optimized to ensure complete strand separation while minimizing DNA degradation. ▪ Effect of Going Out of Normal Range: » Too High Temperature (>98°C): May cause DNA fragmentation, loss of template integrity, and degradation of the polymerase. » Too Low Temperature (Tm+5°C): Reduced primer binding, leading to low or no amplification. » Too Low Temperature (80°C): Reduced enzyme activity, leading to premature polymerase inactivation. » Too Low Temperature (

Dörthe Andrea Kesper Hello, Dörthe Andrea! Thank you very much for your answer!

1. We only know that template is chicken bronchitis vaccine.

2. We can not check the integrity of our template.

3. We have ready-made kits and do not know the specific features of enzyme and kits. It is confidential and the producer do not provide us information of all details.

Today we changed the condition of PCR and I will back with result after.

Ahmed M. Abdelmaguid Hello, Ahmed M. Abdelmaguid! Thank you for your reply, it is very detailed and clear. We will try to change the conditions of PCR and I will be back soon with feedbacks.

Nailia Sabitova

Dear Nailia, After carefully reviewing your qPCR results, there are some critical issues that need to be addressed regarding both runs.

First Run (Image 1): In this run, your amplification curves for the Cy5 channel show strong and clear exponential amplification in multiple wells. However, we noticed that your negative control wells (A03 and A04) also show amplification with Cq values around 31.23 and 31.55, which indicates a false positive. Negative controls (labeled as “Neg Ctrl”) are designed to contain no target DNA and therefore must not show any amplification. Their fluorescence signal should remain flat across all cycles, indicating no DNA amplification occurred. Therefore, this result strongly suggests contamination, either during sample preparation or pipetting.

Second Run (Image 2): Similar observations are seen again. While many of your unknown samples amplified as expected, your negative control well A02 amplified at Cq 14.85, which is even earlier than some positive samples. Such early amplification in a negative control is a strong indication of serious contamination, most likely with a high concentration of template DNA. Furthermore, multiple wells show similar amplification patterns, which suggests the contamination may have spread across different wells or reagents.

What Should Have Happened: Negative control wells should show no amplification at all (flat line at the bottom of the graph). Only the positive control and truly positive samples should show a sigmoidal amplification curve. Cq values in negative controls must be marked as “Undetermined” or N/A, not measurable.

Recommendations: 1. Repeat the qPCR run using fresh reagents and under clean, controlled conditions. 2. Use filter tips to avoid aerosol contamination during pipetting. 3. Prepare your master mix and samples in separate, dedicated areas 4. Always load negative controls first to avoid back-contamination from positive samples.

Hi Nailia,

I have seen that Ahmed M. Abdelmaguid wrote that there might be contamination issues in your experiments. This might be true as you get a signal in your negative controls (however I am not sure which curve is showing your negative control and your threshold is set really low - I can't even see it in the first run - so that you might pick up some backgroud signal. The treshold is usually set in the middle of the log-linear phase).

To avoid template contamination I think the recommendations you got from Ahemd are really good and you should follow them. Working really clean will also diminish the chance that you get any issues with your template. As you work with a vaccine I think you have an mRNA template (probable nucleoside modified). mRNA is ususally really prone to degradation (however I am not sure about nucleoside modified mRNA). If you do the revererse transcription indepently from the realtime PCR I would advice also to repeat this step according to Ahmeds recommendations

However, I don't think that template contamination explains the weird curves from your samples in run one (blue lines) and the missing amplication of your samples in run 2 (blue lines). Template contamination should lead to false positive and/or wrong ct/cq data as your signal might come up to soon but usually does not lead to no amplification at all.

Form me this points to a problem with the PCR itself (and/or the template). Usually, in this cases it is advice to controll you PCR product with gel electrophoresis after the run to see if you have amplified anything at all ( I don't think you have any amplification of your samples in your second run and I don't think that you have proper amplification in the first run). It could be also helpful to test your PCR condition in a normal PCR - however as you have no gel electrophoresis I think this is not feaseable for you.

One reason for problems with the PCR could that your PCR conditions are not optimal your primers. I think you can try the annealing/extension temperatures ranging from >55°C to

The two biggest issues are Mg concentration and amount of primer added. If using a crude method of extraction like alkali, the Mg may be chelated to the extent that its levels are too low for polymerase activity. Primer interactions, due to high concentrations is the other significant factor. By increasing the former and decreasing the latter, it is likely to solve the problem. Also, ASA (allele specific amplification) can increase one amplicon while decreasing the other. To avoid this process the amplicons separate, then combine when running out on a gel. Also, never rule out the machine (a near mistake I made). If the lid temperature is too hot it can impact the level of amplicon to the extent of it being undetectable. Ramp times are also important; they should be minimal between stages.

To be short, there are too many reasons for PCR to fail. Troubleshooting qPCR is difficult, and reading or citing textbooks is not usually helpful. Dear Nailia, my advice to you is to find a good expert in your department/institution.

Great insights here….

One problem I have encountered in our lab which I see most people might not is caused by the arrangement of the samples in your PCR plate machine. Sometimes the blocks don’t sit well resulting in tubes being deformed or at the end low volumes in some tubes and inconsistent in readings of fluorescence. You can also check your tubes at the end and arrange in the best way for example for quantistudio having tubes (empty) on all corners or arranging in the middle.

Hi Nailia,

Sorry to hear about your PCR Problem. There are several factor behind a successful PCR.

Following points may help you.

1. What type of RNA are you looking for? mRNAs or Small RNAs

2. What's the concentration of your cDNA?

3. Check your Primer stats.

4. you can Change the SyBR green.

95 °C for 2–3 min (instead of 15 min) to preserve polymerase activity and prevent enzyme degradation.

Standard Program: 95 °C, 15 s (denaturation) 60 °C, 1 min (extension) Annealing Gradient: Run an annealing temperature gradient from 55 °C to 65 °C in 2 °C increments to determine the optimal primer–template binding temperature.

Prepare reactions with primer concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 pmol/µl. Select the concentration yielding the lowest Cₜ with minimal baseline noise.

If the master mix allows adjustment, test MgCl₂ concentrations from 3 to 6 mM (typical default: 1.5–2.5 mM). Fix the concentration that provides the best balance of amplification efficiency and specificity.

Use Primer-BLAST or Primer3 to confirm: Absence of significant primer–dimer formation. High target specificity with no plausible secondary binding sites.

Measure A₂₆₀/A₂₈₀ ratio (ideal: 1.8–2.0). If purity is suboptimal, further purify or dilute the DNA.

Positive Control: Known-template sample to verify reaction efficiency. No-Template Control (NTC): Water-only reaction to detect contamination.

When using SYBR Green, perform a post-PCR melt-curve to confirm a single peak, indicating a unique amplicon

If amplification is weak, increase to 40–45 cycles, especially when the target is low-copy.

Consult published papers targeting the same gene to adopt proven annealing temperatures and primer concentrations

If problems persist, replace the master mix or polymerase with a fresh lot from a different supplier (check expiration and compatibility

Malak Mohammed Abu Sheleel Asif Mondal Taona Zinyaksa Igor Kutyavin Allan Ackerman Dörthe Andrea Kesper Hello, everyone! Thank you for all your answers, We have decided change the PCR conditions: divided on the two steps - annealing and extension and had good results.