Questions related to Real-Time PCR
I recently conducted cDNA synthesis followed by conventional PCR for quality assessment. In the course of this experiment, I observed some unexpected results that have raised questions about the reliability of my cDNA and its potential impact on real-time PCR experiments.
Specifically, here are the key observations:
- RT- Sample: The reverse transcription negative control (RT-) showed the presence of two bands, with one of them being my target gene. This was unexpected as the RT- control is typically used to confirm the absence of cDNA synthesis. I'm puzzled by the presence of my target in this control (the NTC did not show any bands).
- Sample Variability: In the PCR results, all of my samples indicated my target gene, but I noticed variations in both stringency and intensity of the bands, despite matching the RNA amounts to 1000 ng during cDNA synthesis (The A260/A280 of all the samples were in the range of 1.8-2). This variability across samples is concerning and may affect the reliability of my results (The A260/A280 of all the samples were in the range of 1.8-2).
My primary concern is whether these observations in the cDNA synthesis and conventional PCR could potentially impact the outcomes of my real-time PCR experiments. Real-time PCR requires high precision, and any issues with cDNA quality or PCR variability could affect the accuracy of gene expression quantification.
I would greatly appreciate insights from the research community regarding the possible reasons for these observations and their potential implications for downstream real-time PCR experiments. Your expertise and suggestions on troubleshooting or optimizing this process would be invaluable.
Hi ResearchGate community,
I have been trying to learn more about the optical differences between block-based real-time PCR machines like ABI StepOne versus rotor-based machines such as MIC or RotorGene systems.
I understand that some systems rely on ROX as a passive reference dye while others state that it is optional to incorporate it and others do not need such a factor at all.
My question is if you add this fluorescent dye to your master mix, would it interfere with the detection when it is being amplified using one of the systems that do not need such normalization?
Highly appreciate any insight in this regard.
I am conducting a comparative experiment using real-time PCR to determine the threshold cycle (Ct) values. For one of the gene primers, I have established that the ideal annealing time is 10 seconds. When I increase the annealing time beyond 10 seconds, I observe no amplification. I have tried various standardization methods and have decided to continue with the 10-second annealing time. However, with this setup, I am obtaining Ct values but no visible amplification, possibly due to insufficient elongation time. I am using Applied Biosystems SYBR Green Master Mix.
Given this situation, I am uncertain whether I should conclude the experiment based on the obtained Ct values, considering the absence of visible amplification. Is it okay to proceed with the obtained Ct values? Your insights and guidance regarding this matter would be greatly appreciated. Thank you.
Hello, I am bachelor student. I extract DNA from urine sample using two different DNA extraction kits and detect alpha thalassemia 1, SEA deletion type by real-time PCR. I use the same of pcr mixture and pcr condition but Melt peak aren’t same location. in the picture, blue is dna from kit A, green from kit B, pink is control and red is NTC. the dna from kit A have melting temperature lower than DNA from kit B. So, l’m not sure what happen.
DNA purity affects to shifted or not?
Does anyone have recommendation for this issue?
I got amplification of my genes in the negative control in Real-time PCR experiment although the Ct values were above 30. I setup my reaction again after making new dilutions of PCR reagents, cleaning the workbenches, using unopened autoclaved tips, and cleaning my pipettes with 70% ethanol. I tried to remove every possible source of contamination but still I am getting the same Ct values in my negative control. It is a TaqMan probe-based multiplex PCR reaction where I am amplifying three genes in the same reaction and I am getting amplification of two of these genes in my negative control. Can anyone guide me how to get rid of this amplification?
Hi everyone. I'm making a real-time PCR primer efficiency curve of long non-coding RNA of exosome, but the curves are inverted. In concentrated samples, Cq is higher (above 30) and in more diluted samples we have lower Cq (above 26 and 27). I would like to know if someone has already made an efficiency curve with molecules from exosomes and how it was because I can't find a solution.
Which tools or methods are used to compare the relative gene expression of real-time PCR value other than the Livak method??
I have values of CQ of both gene and internal reference. I want to know which methods compare the gene expression among the samples. The relative expression between samples.
I have used the Livak method. But I want to know other methods or tools.
Please guide me.
Hi, I have NTC contamination in real-time PCR. I checked all suspicious items such as primers, master mix, and distilled water, and also the workspace. but unfortunately, I can not remove the contamination.
I need to do some analysis on my personal computer and unlike the Quantstudio software, I can't find a free version of the 7500 Real-Time PCR software
I'm analyzing the same DNA samples using conventional PCR and real-time PCR (Sybrgreen), with the same PCR parameters and primers. However, while in the qPCR most of the samples are negative, in the conventional PCR they are mostly positive. Does anyone has an idea how to explain that?
We performed qPCR analysis using SybrGreen dye, where the template was gDNA, on the Applied Biosystems 7500 Fast Real-Time PCR System (software ver 2.3). The reaction conditions were optimized using standard PCR conditions (denaturation at 95°C for 60s, annealing at 58°C for 30s, elongation at 72°C for 30s). First, these conditions were tested. We also tested the default program for SybrGreen (2 step, 60°C annealing + melting curve, photo1,2) and many other combinations each time, without obtaining any curves (photo3). I know that the reaction itself, reagents and temperature profile is well optimized because after the reaction, we made an electrophoresis of the product (photo4).
Do anyone have experience with this software? Is this software bug (do you press any extra option?) or do I make something wrong? Additionally, is it possible to preview the amplification plot such that it is presented on a linear and not a logarithmic scale during reaction? I will be grateful for any tips.
My concentration was 123.5 ng/uL
260/280 Ratio - 1.725
260/230 Ratio - 0.401
Can I synthesize cDNA from the above RNA?
Need suggestions please.
I know that in LighterCycler 480 system, we can use Absolute quantification/fixed points methods and then set a reasonable noise band to estimate the CT values.
My workmate told me to use this analysis method in repeating her work, however, the qPCR machine in our lab is Applied Biosystems ViiA ™ 7 Real-Time PCR System. And I really can not find out how to set the noise band or apply this method.
Thank you so much for your attention and advices!
I performed real-time PCR on cDNA of samples that had a nanodrop quantity of 1000+ ng/ul, but when i perform i do not get any proper results. i am attaching the report of my real-time. kindly help me out. should i synthesize the cDNA again or should i change something in the way i'm performing real-time pcr.
I am on the way to order a Real-Time PCR system for following analysis in my lab,
- Relative quantification of gene expression
- Genotyping by HRM
- MicroRNA expression
I‘ve used Rotor Gene Q before, but I need plate-based system. So I would like to switch Applied Biosystems devices.
I am confused between Stepone plus and Quantstudio 3 instruments.
I need your recommendations with reasons.
P.S. I am a Mac user. Software compatibility is important.
I'm new to Real-Time PCR and I'd like your insight on this. I have generated standard curve by doing 1:10 serial dilution in triplicate. However, the result obtained, of which R value is to determine the efficiency of the assay is poor (R> 114%) and there are only three points out of 10 that fall on the line. Should I rerun test on the rest of the deviated points or repeat the whole procedure again?
Hi, I conducted qPCR using probe included FAM and BHQ-1.
At two annealing temperatures(60 and 65 C), the Ct value, R^2 and PCR efficiency in the amplification plot were also within the normal range.
When the PCR product was confirmed on an agarose gel, a normal band was confirmed.
However, only a slightly higher level of fluorescence than background was detected in the multicomponent plot(square box in figure).
But why is the Ct value calculated? Can I continue to use this probe?
In the same experiment, graphs for other probes containing FAM were normally generated.
I used a KOD DNA polymerase for PCR that works very well. Then I used this enzyme to run a TaqMan probe real-time PCR but the results waere unexpected. I attached the results. The fluorescence rise very fast during initial cycles and it reach to a plateau much faster than the control. The control enzyme is a Hot-start but the KOD is not. Can this be the cause of this phenomenon or is there another reason involved?
Red: Control enzyme (Hot-start)
Blue, green, purple, and pink: KOD with different buffer.
What would happen if my primer pair can amplify several genes? (ie. it is not specific to only one gene) Would it be the cause of false positive results?
1. I have BLAST my primer pair and it can bind only my bacterial species of interest.
2. I use a specific nucleotide probe for fluorescence detection in real-time PCR.
which genes can I check in real-time PCR in cardiac fibrosis? Has anyone done it? I would appreciate it if you could help me.
I have made real time pcr with B-ACTIN and TNF-ALPHA . It give ct values with B actin but not give any values with TNF-alpha.
I use 25microlitre pcr
1.50 microlitre primer
3 microlitre cdna
7.50 microlitre cybergreen
13 microlitre water free
I'm passionate in oncology research. I did bachelor in Zoology and now pursuing MPhil Molecular Biology and Biotechnology and working on cancer genetics. I am greatly interested to do PhD in cancer research from a world renowned institute but I think with this profile I would get a position in top ranked institute for PhD. Should I go for another master from a renowned foreign institute with major in oncology?
Hello, our Real-Time PCR system (AB7500) is acting up with the status constantly showing “Waiting for the heated cover to reach temperature” when we start the run. Neither the cover, nor the block are getting heated. Can anyone suggest what the error might be and how this might be fixed?
Thanks in advance!
Of course, the samples' Cts do not fall within the standard curve.
Just curious about the scientific community's impressions on this topic because we recently discussed it in our lab.
The expression of nirS and narH genes involved in the nitrite reductase and nitrate reductase pathways of Thiobacillus denitrificans ATCC 23644 treated with zero-valent iron nanoparticles was measured by Real-time PCR based on SYBR-Green I fluorescence, which showed a significant increase compared to the control group.
I performed a Real-time PCR on colon cancer cell lines after treatment with some anti-cancer agents. Although the expressions of caspase 8, 9 and P53 are increased but the Bcl2 expression is increased as well. How is it possible? because Bcl2 is an anti-apoptotic protein.
I am almost a newbie in the miRNA world :)
Try to identify miRNA in heart tissue samples according to a TaqMan™ Small RNA Assays user guide (No 4364031). I use Taqman chemistry, 7500 Fast Real-time PCR (Applied Biosystems).
And it didn`t work (please, see attached pics).
Do you have ideas why?
First pic. - RNA was not standardized by concentration, second - 5 ng of total RNA was added to RT mix. And things only went worse ((
I use TaqMan MicroRNA RT Kit, Universal PCR Master Mix, MicroRNA Assays (U6B, miRNA-1, -34a, 320 - all "hsa").
I would be very grateful for your help!
Have a good day!
Glory to Ukraine! Glory to the Heroes!
i have been developed a taq man real time pcr for my work… but i have a problem in maintaining my STANDARDs and after a week the load of plasmids gets low… how can i solve this problem?
I do use multiplex qPCR, I use 4 different templates, Among these samples, only one sample give me one good Peak in flouresent per cycle and ct, everything is the same but the type of template. Does anyone could help me with this kind of abnormal peaks.
I am looking for to buy a RT-PCR machine-96 wells. Please mention your experience with the machine in your lab. Any pros and cons. Thanks
Dear ResearchGate community,
I am working on DNA samples obtained from patients screened for cervical cancer/ HPV infection. To calculate the viral load of each sample, I am looking for a technique to know the amount of the viral DNA present in each of the samples.
That said, considering the fact that the viral genetic content can be present both as episomes as well as integrated into the human genome, what would be the best approach to get insight from the "effective" viral load?
In other words, for the diagnostic purposes which one of the two types of viral DNA- if not both- is of significance, and how can it be quantified?
Many thanks in advance,
I have loaded my qPCR products onto a 2% agarose gel and ran them at 60V for 1 hour. I ran 2.5ng of cDNA in a 10uL qPCR reaction. As you can see in my image there is smearing of all bands in the heavy direction. I have tried reducing the amount of sample loaded and switching to TBE buffer and this helped slightly but still gives the smearing that is shown in the attached image. What else can cause this?
So I got these weird amplification curves in SYBR green qPCR using cDNA extracted mouse tissues. The miRNA was retrotranscripted with stem-loop primers. The curves are weird because the fluorescence was not uniform at the start of the reaction, increased suddenly and dropped after reaching plateau. The weirdest part is that the anomalies did not happen in every well, but only some of them. The melting curves showed one main peak and much fluctuation. However, another reaction done using the same RNA material and kit showed no anomaly. Sincere thanks in advance for your advice.
I understand that the fragment size is a limiting factor, but I need to test and know what I can adjust in the cycle.
I have 2 groups in my study (treatment & control). I did taqman gene expression assays for the gene of interest (GOI) and a house keeping gene for each individual in the study Post treatment. I don’t have baseline values for any of the individuals. Now I have Ct values for both GOI & housekeeping gene for each individual in treatment & control groups. Can anyone help me in the next step?
I think the delta delta Ct method (Livak method) cannot be applied for my data as they are 2 different groups, not pre and post-treatment data. What can I use alternatively?
Thanks in advance.
While Working in molecular biology Lab, QuantStudio™ 3 Real-Time PCR System, 96-well, 0.2 mL, laptop is our tool.
I want to calculate the Genomic equivalence, then want to calculate it for parasite number per micro liter of blood.
Hence, I am looking advises from any one working on this area is very helpful.
What are the limitations and disadvantages of Real-Time PCR (RT-PCR)?
What is a more specific and sensitive technique that can be used in the laboratory instead, particularly in cancer diagnosis?
If you take a look at the image, the upper virus X assay (for Rotavirus) is giving off a lot of fluorescence at the end of the run (circled) which is clearly not real (we know these are Rota negative samples), this is affecting the baseline setting making the positive samples look like they are giving off poor signal.
The lower figure for the virus Y assay (for Norovirus) shows what the assay should look like but is just an example targeting a different virus
Is this primer/probe degradation and if so why don`t I ever see this is any of my other assays
I'm studying on 5 miRNAs under chilling stress in tomato plants. I would like to know if the concentration of the cDNA used for first stand sample should be the same as the concentration of cDNA used for the main samples on the plate? For example if we have diluted our cDNA 3 fold for the main samples, should we dilute the cDNA that is used for the first standard sample? Should I dilute a diluted cDNA 4 times to prepare 1:10, 1:100, 1:1000, 1:10000 standard samples? Or I should use undiluted cDNA initially ? Thanks for your time and consideration
In the kit guide, it is mentioned that it is recommended to dilute the cDNA 3 fold before making a real-time PCR reaction setup.
Can I add 20 ul of nuclease-free water to 10 ul of cDNA to dilute 3 fold? Or I should add 90 ul of water and then I should take 10 ul of the final solution and add 90 ul of water to it again to make a 3-fold diluted cDNA?
Moreover, I am going to dilute cDNA for standard mix synthesis to use them in the real-time plate vessels. (S1,S2,S3,S4,55). I would like to know for diluting 1:10, 1:100, 1:1000, and 1:10000 of cDNA should I do serial dilutions?
Is there any database to identifies which protein isoform is expressed in a particular tissue?
I want to design primers for real-time PCR on cartilage samples. I need to be sure which mRNA variants are specific for cartilage.
We have a recurring problem in our routine.
After RNA extraction, we performed reverse transcription of the samples and a negative control with water (Blank). We proceeded with a pre-amplification from this cDNA, including the Blank. We consecutively do the real-time PCR of the pre-amplified samples together with the Blank that "run" along, in addition to using as well as water instead of the sample.
What is happening is that Blank is amplifying (in late cycles), in which case there is no sample in the reaction, so it was not meant to amplify. We have already tested reagent by reagent. That well that we make with water in real time does not amplify, so we discard water contamination.
Do you have any idea what it could be? Have you been through this?
I am putting together a standard curve where the concentration of the plasmid is 0.167ug/ul, plasmid bp is 5821, and gene of interest is 1790 bp.
For the plasmid I get : 2.618×1010 copies
For GOI: 8.513×1010 Copies
My question is should I use the plasmid or GOI copy # when developing my standard curve?
I've used this site for my calculation
Copy number calculator for realtime PCR | Science Primer
Normally Realtime PCR also known as quantitative PCR (qPCR) is used for investigation gene expression, in which case Reverse Trasncriptase-PCR is used,starting from RNA. Whenever realtime PCR is discuessed, first thing that comes into mind is gene expresseion. My question is what is its role in detection of a mutation? Is it the right thing to use it for mutation detection starting the reaction with genomic DNA? I have done an experiment where I have used qPCR technique for detection of a mutation in whole blood. Genomic DNA was used and primer sets targeting the wild type and mutant sequence were used.
I would like to study correlation between four transcripts (fold changes of mRNA expression) at different time intervals (5 time points). How can I perform this analysis?
Suppose, I have a treatment group and a control group. I have measured the relative expression (delta delta ct) of two genes by real-time PCR analysis. Both the two genes are analysed from the same samples and the same reference gene was used. In this case, if one gene has higher relative expression than another gene, can I say that gene is highly expressed than the other gene. My question is if the relative mRNA expression from two genes can be compared? another question is, is it ok to produce a heatmap from relative expression data of different genes?
I'm using degenerate primers for both Real-time and PCR. Although these degenerate primers work well in Real-time PCR, I can not detect the 100 bp size bond that I'm looking forward it in my Gel electrophoresis after PCR. Does anyone have been faced with such a problem? Any suggestion?
Thank you for your attention and time.
I want to use an internal control for a real-time PCR method for the detection of DNA viruses. This control would allow me to detect PCR inhibitors. The protocol is based on TaqMan chemistry. I have the primers and the probes, but I don't know how to develop an internal control. It would be really great if someone could help me with this.
I did Real-time PCR for some miRNAs for (two groups ((responders and non-responders)) to chemotherapy). I had the values of CT values, delta CT, delta delta CT values and finally fold change. I want to do a ROC Curve for them to determine the sensitivity, specificity, PPV, NPV, AUC, and a cut-off value for the test for each marker.
I am using Graphpad prism 7: (Analyze> column analyses> ROC Curve).
I want to know if we put CT values for each marker or fold change and why? Is there any data I have to put beside real-time data?
Moreover, I want to do ROC Curve for combined miRs after the individual ones. How can i do this?
I am currently studying the effect of a particular drug on the expression of a number of genes in the hypothalamic tissue in a number of samples.
After I designed the primers, taking into account the necessary standards, I achieved BLAST for them.
When I made several set up Real-time PCR runs I had a problem of getting either one peak (product), two Peaks (product and Dimer-primer), or one Peak (Dimer-primer) with different samples
Although I raised and decreased both Tm, primers, and cDNA concentration without solving this problem.
In addition, the b-actin primer gave me one peak (product) without any problem with all samples
Could the expression level of the studied genes be related to the extent of the appearance of these three cases?
It seems to me like the 2009 method is preferable, but many recent papers use the 2002 protocol. Is there any reason for this?
When performing Viral RNA extractions I perform a negative extraction using water. This is then run as a negative control for my RT-PCR reaction. Recently this control has been showing up RNAse P positive. In addition, when I run the same extract negative sample in duplicate on my PCR plate, sometimes I get RNase P in only one of the duplicates. What is happening? It only ever happens in the Extraction Negative, it never occurs in my PCR negative control.
This issue is occurring for multiple technicians.
Hi, I'm doing presence/absence experiments with a comercial kit qPCR. The software requires an IPC-blocking agent but the kit does not have. Is there any configuration that allows it without IPC blocking agent? Moreover, the presence/absence graph does not appear in the analysis.Who know how to fix this problem? Thanks.
How can we use traditional pcr to detect corona? Is there an acceptable method of the World Health Organization? What is the last CT obtained from the real-time PCR method that can be seen on agarose gel electrophoresis ?
I have performed high-resolution melt analysis using KAPA HRM FAST mastermix; PCR conditions were the same for both experiments - Image 1 and Image 2. However, there is a lot of variability in the two consecutive experiments. I haven't changed any parameter between the two experiments. (Image 2 contains additional samples).
Can anyone point out what I could be the possible reasons for variation in results.
I performed a real-time PCR analysis on cDNA samples deriving from one microalgal species grown under different conditions.
In order to adjust the expression-fold changes on the base of the amplification efficiency, I used one condition (control) to prepare serial dilutions and obtained a calibration curve for each target gene.
I do not know yet if the type of sample significantly influences the efficiency (for a temporary shortage of reagents) but from what I read usually researchers focus just on efficiency variation depending on the primer pairs.
Is this enough or I should do the same also for the rest of the conditions? And if yes, how the Pfaffl formula would change in order to consider different efficiencies per target AND per condition?
Which Real-Time PCR Instrument is better? Those that work with passive reference dye or those that do not work with passive reference dye.
I am exploring a number of SNPs, unfortunately although the test are ok, I am perplexed because my preliminary results are nearly all against what is reported in the literature.
To confirm my workings, I need to understand if I have done this correctly.
I am using prepared primers from TaqMan for a realtime PCR. This uses two dyes: VIC and FAM, each associated to one allele.
An example of a primer would be the following:
where the manaul stated that A should be VIC and G would be FAM.
Does this mean that those results with a low Ct on VIC and a high Ct on FAM would be A or G allele?
I've been performing qPCR on an ABI "StepOnePlus" machine for the past year. I produce RNA either by kit or Trizol, perform RT using Quantabio "qScript cDNA synthesis kit", and perform SYBR green qPCR with Quantabio "perfecta sybr green fastmix rox".
I've recently realized that I am not producing standard curves with good slopes anymore. This is true for new primers I ordered based on literature that are supposed to be perfect, as well as for all my most reliable primer sets that I've previously validated (they used to have good R, Good 3.3-/+0.3 slopes, only one melt curve product, no primer dimers or products in NTC wells).
All my curves now shift between 55-and-75% efficincy instead of >90%, in spite of my R^2 values staying great and the melt curves still showing single products.