Real-Time PCR - Science topic

Ritu Kumari is correct, the first thing to check is that you are amplifying only one PCR product.

But if all of your controls are working, and you know it's just one molecule, and you consistently see a double peak for ALL of your samples with that particular pair of primers, see my explanation below.

How large is your amplicon? The longer the DNA molecule, the more likely that it will melt in "regions" based on GC content. I've seen it when using melt-curve based genotyping. Anything over ~120 bp or DNA with regions of very different %GC will often get a messy peak.

The following R-package could provide useful if you decide on using any of the following tests: two-proportions z-test, a two-group comparison of Poisson data,comparison of negative binomial data, one-sample or two-sample t-test, t-test for non-zerocorrelation, Fisher’s exact test, signed rank test, sign test, rank sum test, ANOVA, andsingle-predictor Cox regression (PDF) Computing Power and Sample Size for the False Discovery Rate in Multiple Applications. Available from: https://www.researchgate.net/publication/378807566_Computing_Power_and_Sample_Size_for_the_False_Discovery_Rate_in_Multiple_Applications [accessed Apr 25 2024].

Dear ResearchGate community,

I'm somehow referring to the same question, I was wondering if it is fine to use a Sybr green master mix containing ROX for a machine that does not require ROX addition, The CFX96 C1000 touch from Biorad will this addition affect the signal detection and if yes are they any ways to subtract it? looking forward to your insights.

Yes, it is recommended to include an internal control in your TaqMan probe-based Real-time PCR assay to monitor the performance of the assay, detect potential issues, and ensure the reliability of the results. You can either design the internal control to work in a multiplex PCR with your target DNA or run separate reactions for the target DNA and the internal control.

good luck,

Djihad Chenna

Hello,

Titration of mixed solutions containing nitric acid (HNO3) and sulfuric acid (H2SO4) can be challenging due to the complex chemistry involved. However, there are methods available to accurately determine the concentrations of these acids in a mixture. One commonly used technique is the "back titration" method, which involves a series of chemical reactions to determine the acid concentrations step by step.

Here is a detailed and orderly explanation of the back titration method for titrating HNO3-H2SO4 mixtures:

This back titration method allows you to determine the concentrations of both HNO3 and H2SO4 in the mixture. It is important to perform accurate measurements and titrations to obtain reliable results. Additionally, using standardized solutions and appropriate laboratory techniques is essential for the success of this titration method.

I hope this explanation helps you understand the procedure for titrating HNO3-H2SO4 mixtures. If you have any further questions or need clarification on any aspect of this method, please feel free to ask.

With this protocol list, we might find more ways to solve this problem.

not exactly.

You are welcome

Lets look at definitions and applications of the methods in your question:

The western blot (also known as western blotting), is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract (or to detect a specific protein in a blood sample for instance).

It is noteworthy to mention that ELISA is a more simple and faster procedure than Western blotting, which is less specific. Western Blotting is a highly successful testing method for confirming positive results from ELISA tests. It is also used as a confirmatory test as it is difficult to perform and requires a high skill level.

On the other hand, real-time polymerase chain reaction (PCR) is a technique of molecular biology based on the polymerase chain reaction. It monitors the amplification of a targeted DNA molecule during the procedure (in real time), not at its end, as is the case in the conventional PCR. Real-time polymerase chain reaction can be used quantitatively and semi-quantitatively.

Hi Erika Perez, I’m experiencing the same issue. Have you found a way to solve it?

Hi Parisa,

As per my understanding low or high RNA purity and concentration ratios can interfere with reverse transcription and PCR amplification, leading to inaccurate results. Clean up your RNA before performing real-time PCR.

Best of luck!!

Thanks for the update. Under optimum conditions ("perfect" amplification efficiency), the Ct should shift by about 3.3 cycles per 10-fold dilution (=log₂(10)), hence about 6.6 cycles per 100-fold dilution. The shift will be larger under suboptimal conditions). If your normal samples have Ct-calues of 24 or larger, then it should not happen that 100-fold dilutions of these samples have Ct-values of less than 30 (you mentioned 27). This is strange.

Apart from this I don't see anything obvious that would possibly explain your findings. Does this happen only for a specific primer/assay or do you see this also for other primers? If yes, it might be due to the instrument. If only a particular set of primers/assay doesn't work well you might just design and order (sligtly) different primers, what might solve your problem.

The sample preparation for real-time PCR on the Illumina Eco system does follow the same general principles as standard real-time PCR. However, there are some specific requirements to adapt the protocol for the Eco system:

- Reaction volume is very small - only 10-20 μL per well compared to 20-50 μL for standard PCR plates. So reagents need to be scaled down accordingly.

- Special 384-well plates are used. Make sure to use compatible optical plates provided by Illumina.

- More concentrated primer stocks may be needed to accommodate the smaller reaction volumes. 100 μM primer stocks are common.

- Always include positive and negative controls.

The actual real-time PCR reagents (Taq polymerase, buffers, dNTPs) and cycling conditions can remain the same as standard PCR. Just the preparation needs to be adapted for the smaller 384-well Eco plates.

Hi Robert, Thanks for the kind reply.

No, you can not use Ct values that don't indicate that your gene-of-interest was amplified.

Something is not working correctly. Either you have primer dimers, your qPCR product is too large, you have contamination, the threshold is set too low in your analysis, or some combination of the above.

What do you see on your standard curve and your positive controls?

Woratit Intap

Many thanks for taking the time to give me more details.

Francesc Codony's suggestion might make the situation clearer, I guess.

Best,

N

Yep, it can be done. Though most of our experience has been on the Minion, I know we've done some work on the Flongle too.

· Cross-contamination: Take care to prevent cross-contamination between samples by using separate equipment and workspace for the negative control. Double-check that pipettes and consumables are not shared.

· Contaminated reagents: To minimize the risk of contamination, handle reagents with caution. Use fresh reagent stocks from a different batch, store them properly, and ensure they are not compromised.

· Aerosol contamination: Employ filtered pipette tips, work in a clean environment, and keep PCR tubes or plates covered while performing actions like opening tubes, vertexing, or pipetting to minimize the potential for airborne DNA fragment contamination.

· Carryover contamination: Thoroughly clean and decontaminate laboratory equipment, including pipettes, tubes, and the thermal cycler, to eliminate any residual DNA from previous experiments that could contribute to carryover contamination.

1. Repeat the experiment using new reagents, preferably from a different batch, to eliminate the possibility of contaminated reagents.

2. Set up a fresh negative control in a separate area using different pipettes and consumables to reduce the risk of cross-contamination.

3. Conduct additional negative controls using sterile water instead of template DNA to determine if the issue persists. This will help identify if the contamination originates from the reagents or the laboratory environment.

4. Ensure that PCR tubes or plates are properly sealed to prevent aerosol contamination during the experiment.

Hi Elham Sheykhsaran

I heard Qiagen has a kit. I was reading about it last week. Please check the files attached.

Thanks

thank you

Hi Suellen,

I'd suggest you look at your trace data. I believe you'll find the curves are not at the quality as required to draw quantitative results from a standard curve. I'd suggest you look into the following points which may or may not apply to your work (as there are many variabilities/requirements of qPCR):

1. Use a stable DNA source of control within a vector

2. Dilute your controls and samples to be a maximum of 10% of your qPCR reaction

I've worked with EV RNA/DNA many times and have not had any issues differing from the norm when it comes to qPCR. I'd suggest you perhaps chat with someone from your group/laboratory and go over the quality of your sample/methodology to confirm.

Good luck.

Hello! I like to use the "qpcR" R software to fit curves, but it isn't necessary. you can use the "delta delta Ct" method. Here is the gold standard instructions for qPCR experiments. Good Luck! https://www.gene-quantification.de/national-measurement-system-qpcr-guide.pdf

Hi Zeynab,

For such issues, you can simply contact Dr. Shanehbandi at Tabriz Medical University, an expert in PCR and troubleshooting. 09144160144

All the best,

Daniel

Check out the scale on the Y-axis on both graphs. You have minimal amplification from any of the samples, that's why the data look so messy.

Start with just a positive & negative control. Once you get those working, then add in the standard curve samples.

Good luck!

There are several reasons why conventional PCR and real-time PCR (qPCR) can yield different results, even when using the same PCR parameters and primers. Here are some possible explanations:

Several factors can contribute to different results in conventional PCR and real-time PCR, including differences in sensitivity, contamination, PCR inhibitors, primer specificity, and reaction conditions. It may be helpful to optimize the PCR conditions and controls to minimize these sources of variation and improve the accuracy and reproducibility of the results.

These video playlists might be helpful to you:

This could happen if the machine is reading the ROX value from your ctr.

You need to go to "set up" then "assign targets to wells", for the endogenous ctr. select "none" in the reference. Then click on "analyze". Your amplification curves should appear.

If again no amplification, then you need to increase the amount of your starting cDNA samples or increase the number of cycles to 45

+

choose the delta-delta-CT option from your second picture as mentioned by Yi-Chun Cheng

For cDNA synthesis, high-quality RNA with an A260/A280 ratio of 1.8 to 2.2 is ideal, and a minimum concentration of 1 μg/µL is usually recommended. For Real-Time PCR, the optimal RNA concentration is highly dependent on the gene of interest, and can range from 1 ng/µL to 500 ng/µL.

Oh I finally figured out it, thank you so much.

Real-Time PCR (Probe type) melt peak factor, also known as the Tm (melting temperature) or Tm peak, is a measure of the stability of a double-stranded DNA molecule. It is the temperature at which half of the double-stranded DNA dissociates into single strands. In real-time PCR, the Tm peak factor is used to determine the specificity of the PCR reaction.

The Tm peak factor is used to determine the specificity of the PCR reaction because it is affected by the length and nucleotide sequence of the DNA fragment, as well as the buffer conditions and the presence of other DNA sequences in the reaction mixture. A specific and efficient PCR reaction will produce a single, sharp Tm peak, while a non-specific reaction will produce multiple, broad Tm peaks. The Tm peak factor is therefore a useful tool for assessing the specificity and efficiency of real-time PCR reactions.

Real-time polymerase chain reaction (real-time PCR) is a widely used method for amplifying and quantifying short DNA fragments, typically less than 200 base pairs (bp) in length. This method is based on the simultaneous amplification and detection of DNA during the PCR reaction, and it is commonly used in a variety of applications, including gene expression analysis, pathogen detection, and genetic typing.

Products larger than 200 bp are typically not amplified effectively in real-time PCR for several reasons:

For these reasons, it is generally not recommended to use real-time PCR to amplify products larger than 200 bp. If longer fragments need to be amplified, alternative methods such as standard PCR or long-range PCR may be more appropriate.

for alignment search? https://blast.ncbi.nlm.nih.gov/Blast.cgi

If the power went out DURING your cDNA synthesis, then the most likely explanation is that you didn't synthesis any/enough cDNA.

The nano drop will only tell you the concentration of nucleic acids - not the type of nucleotide, length of molecules etc.

Try again and good luck!

Applied doesn't have a native mac version so for both you will have to use a virtual windows machine on your computer.

It have been a while but BioRad used to have a mac version for their qPCR software and I quite liked the CFX when I worked with it (nearly 10 years ago).

You'll have to repeat all of it.

But it's normal to have limits of your useful detection range. Too few copies and you don't get efficient amplification. Same can happen for too many copies of the template.

The R-square value tells you have precise & accurate repeatability for technical replicates.

The slope of the Ct vs template concentration tells you the reaction efficiency.

Both of these measures are important.

Good luck!

Is it probe degradation? If the probes degrade, and the quencher probe separates from the reporter, it will cause phosphorescence that increases linearly instead of exponentially. Remember that automatically these graphs are shown on a log scale. I do not believe there is a true asymptote in your left hand plots (directed to OP). You can linearize the graph (change axis so it is not a log scale) and see if the growth is exponential or linear. If it is not exponential, it is not caused by amplification but rather by probe degradation.

To OP: It's hard to read your axes, but it seams your c(t) is very different for your plot on left and plot on right. If you made them the same, and scaled them the same, the two graphs would not look the same (I think, if I'm deciphering the axes correctly, but hard to read). If you placed your C(t) at 0.1, the plot on left would look very unreliable, the plateau being far too low.

Hi

Yes, I also agree you need to optimize the reactions first, what ever polymerase you are using. Also the nature of enzyme (hot start/regular) will also make difference.

On top of what Aissa Saidi said, I would say that it could also effect sensitivity as if the primer for target is being producing other amplicons. The test will not be able to have enough primers/dNTPs etc for your target of interest if you have templates competing for the primer. Hence, even if your the target template was there, and you had some primer-target interaction, the non-specific amplicons could out compete the target of interest amplicons so you would always have sensitivity issues in this case.

Pankaj Kumar Thanks a world

Hi,

3 microlitre cdna, this is too much.. I would suggest to check the Ct values for B-actin first. Check your primers too.

I am also at a similar position as you are in. I have completed my MSc. in Genomic Medicine from the University of Birmingham. I am planning to learn Bioinformatics, maybe I will do that from 6months courses and then try to gain some experience in this field.

I think gaining some practical experience is quite necessary at the moment for you as well. After that you can always opt for PhD.

Thanks a lot for your answer

Better check your service contract to see if your machine is under warranty. You will not be able to fix this yourself.

Good luck.

ct values above 40 are not considered as the fine category values to calculate the relative mRNA expressions. usually, when we set the qPCR, the maximum cycle we set is 40. In order to get the desired cycle ratio; one way to do this is by increasing the DNA concentration.

This step is vital to double check of the absence of primer dimer and the specificity of the used primers. In addition to avoid the background noise of the dye used. Therefore, it has become an important step particularly when using SYBR Green I. Thus, it has been recommended to conduct a melting curve analysis.

Dear Solmaz Morovati

No problem. Happy to be.

Best reagrds

AB Bayazid

Saurabh Mandal you were absolutely right. For unexplained reason,the TaqMan master mix didn`t work! We used another and got results.

Thanks!

It may be that your dna is absorbing onto the plastic of the tube or it is being degraded by nucleases or acid pH.

You can store lyophilised and make up in TE just before the assay or add some non amplifying dna or rna to your standards before storage. The cold dna will block the absorption sites on the plastic tubes. Siliconisation of the storage tubes may also help

Hello dear Ali

PCR for detection

and

q-PCR for gene expression rate.

As far as I know abnormal peak like represent Noise. This may be due to any contamination or your template is not pure due to which you are not getting desired peaks. usually, you see this kind of peaks before the reaction reaches ct value.

As you are using multiple primers have you checked for the formation of dimers?

Lightcycler (Roche) outperformes CFX.

Why don't you run Real-Time PCR (LightCycler)? It can be used for quantitative analysis of a gene copy number so it can be used for viruses as well.

You can quantify your obtained DNA by checking the concentrations. Concentrations lower than 1.7 might not give the desired bands.

You should contact tech support for your machine. Looks like an error in the hardware or software.

Also, make sure that your primers are annealing to exons, not introns, or you won't get amplification from cDNA.

You can change the time & temperature for all 3 steps (denature, anneal, extend).

As Laura Morais Nascimento Silva says, check the ideal cycling conditions for your qPCR mix. Good place to start.

Not all primer pairs are efficient enough for qPCR, but in my experience, the primers that give a bright band at a range of annealing temps are ones that are more likely to work.

Good luck!

Manar Serageldin The two most common methods for analyzing qPCR data are double delta Ct analysis and the relative standard curve approach (Pfaffl method). Because both approaches make assumptions and have limits, the method you should employ for your analysis will be determined by the experimental design.

Take a look:

1. https://www.youtube.com/watch?v=GQOnX1-SUrI

2. https://www.youtube.com/watch?v=y8tHiH0BzGY

The best way to do this is to prepare a sample of parasite DNA from a known number of parasites, make a dilution series and then use this as a standard curve. If you cannot obtain such a sample, there are ways round the problem. I work with pathogens that cannot be cultivated in cell-free media and which are difficult or impossible to enumerate. To prepare my standards, I use a plasmid containing the target sequence (several suppliers can produce such plasmids using a synthetic sequence; I use Genscript). DNA concentration can be converted to number of copies by dividing by the mass of the plasmid. Then you make a dilution for use in the standard curve.

The problem with this approach is that you have to know (a) the number of copies of the target sequence in the parasite genome and (b) the number of cells in a parasite. If you don't know this, and you cannot get an accurately counted sample of parasites, then absolute quantitation is essentially impossible.

Hello Mohammed Fatih Rasul

The limitations of Real-Time PCR are as follows:

1. High false-negative due to presence of amplification inhibitors. This holds true for pathogen detection particularly, for new emerging or highly variable pathogen.

2. The multiplexing is still limited in Real-time PCR.

3. The variation increases with the number of cycles.

4. The overlap of emission spectra.

5. The occurrence of non-specific binding particularly, when carrying out SYBR green analysis.

6. The PCR product increases exponentially but one cannot monitor the amplicon size.

7. Kits are not available for all kinds of genes and disorders.

8. It needs specialized laboratories, operated by highly trained technicians for developing novel qPCR assays, which makes it too costly and difficult to scale up.

You could use other techniques that could be performed in the laboratory.

1) Fluorescence In Situ Hybridization (FISH), also known as molecular cytogenetic testing, is a way to visualize and document the location of genetic material, including specific genes or DNA sequences within genes. FISH is used to look for the presence, absence, relative positioning, and/or number of specific DNA segments under a fluorescence microscope. FISH is particularly helpful in identifying copy number variations, especially translocation and amplifications.

2) Comparative genomic hybridization (CGH) is a special FISH technique (dual probes) which is applied for detecting all genomic imbalances. CGH is used to determine copy number alterations of genome in cancer and those cells whose karyotype is hard or impossible to prepare or analyze.

3) Single Strand Conformational Polymorphism (SSCP) is one of the simplest screening techniques used for detecting unknown mutations (microlesions) such as unknown single-base substitutions, small deletions, small insertions, or micro inversions. A DNA variation causes alterations in the conformation of denatured DNA fragments during migration within gel electrophoresis. The logic is comparison of the altered migration of denatured wild-type and mutant fragments during gel electrophoresis.

4) Heteroduplex analysis in which a mixture of wild-type and mutant DNA molecules is denatured and renatured to produce heteroduplices. Homoduplices and heteroduplices show different electrophoretic mobilities through nondenaturing polyacrylamide gels.

5) Denaturing Gradient Gel Electrophoresis (DGGE) has been used for screening of unknown point mutations. It is based on differences in the melting behavior of small DNA fragments (200-700 bp); even a single base substitution can cause such a difference.

6) DNA microarrays can detect thousands of genes at once. DNA “chips” or microarrays can be used to test for multiple mutations. In this technology, single DNA strands including sequences of different targets are fixed to a solid support in an array format. On the other hand, the sample DNA or cDNA labeled with fluorescent dyes is hybridized to the chip. Then using a laser system, the presence of fluorescence is checked; the sequences and their quantities in the sample are determined.

7) Use of Next Generation Sequencing (NGS) allows comprehensive description of germ-line DNA, analysis of somatic mutations and RNA profiles in naturally occurring tumors, systematic analysis of microbiomes, etc. The resulting data can help in the identification of novel hereditary syndromes, molecular targets for cancer therapy, tumor-specific diagnostic markers, etc. But this technique would be expensive to work with.

Best Wishes.

What methods you use depend on factors like how many samples and the distance between the snps, how many negative results you expect and whether you will end up sequencing for confirmation after finding the snp. So if your snps are within a single sequencing amplimer and samples are few and the changes are common then sequencing from the start may be good. If you have many samples to test but few at the end to sequence then ARMS screening followed by sequencing interesting detected samples works

I work in Covid laboratory and for 2 years we barely experienced this kind of curves (to be precise 4-5 times in total). However, recently, we faced this late raise curve that 99% appears with one gene (E gene or N gene). During the last two months (Jan and Feb 2022) we faced an outbreak with new COVID variant Omicron. therefore with had to verify which curves are real presumptive and this added to our work load greatly. Some people here asked if someone tried using different extraction method or changing lot number so I am writing this to answer those with these Qs, in case they have not solve it yet. In one case it was primer probe problem. We changed the lot number and the problem solved (see the attached foto). In other cases we found out it happens randomly and doesn't follow any logic. We changed extraction method and we also compared a set of samples with two different extraction methods and saw the curve happened randomly in both conditions.

Briefly, I think it is more probe degradation issue than anything else. Keep in mind Ct under 40 is consider presumptive and can be the onset of infection. So in our lab, we put under investigation all the late raises with one gene and will be re-swab 24-48 hrs later. Statically, we found out around 10 percent of cases turned presumptive with further investigation.

I hope this help. Also if anybody has input on this subject I would be happy to hear your suggestion.

PS.: attached foto is an example of a probe lot number that was not good (degraded probe) in black circle and a random late raise after 35 cycle that only appreared in one gene (N1 gene) circled in blue. The red is positive control gene.

Melisa Ermert Flor Torres-Juarez Umadevi Subramanian

No, you CAN'T extrapolate for qPCR data. Your data points have to be within the linear range of your curve (interpolated).

As Leonid Ilchuk said, you'll have to figure out empirically if any dilution is needed for your particular genes of interest in your samples.

take your 10 μL of the stock solution and dilute with diluent to a final volume of exactly 30 μL( add 20 ul of diluent). This will yield 30 μL of diluted solution and results in a final dilution factor of 3. This represents a 3-fold dilution. It may also be said that the stock solution has been diluted 3 fold.

Hmm, hard to say...

It seems that isoform uc002bna.2 (http://genome.ucsc.edu/cgi-bin/hgGene?db=hg19&hgg_gene=uc002bna.2&org=human) is expressed in most of the tissue types (see the image in the attachment) ... but there are no data for cartilage tissue type specifically

So you may risk it and proceed further with primer design, or you can try to find expression of your isoform in some cartilage-specific public RNA-seq data (laborious)

Martin

Are you running a melt curve at the end (if not, you really should).

"Amplifying late cycles" is a hallmark of primer dimers/mispriming: since the only real difference between your water control and your "blank" is that your blank also contains all the RT reagents (including the primers for that reaction) and the primers used for pre-amplification, I would ascribe "primer dimers" as the most likely source, here. The more primers you mix together, the greater the chances two of them will anneal to each other slightly, and it only takes one aberrant binding event to create a viable amplicon for subsequent cycles.

A melt curve helps distinguish real amplicons (melt peak ~78-85, identical for all wells) from primer dimers (melt peak ~70-75, usually fairly broad), and from mispriming (lots of peaks, messy trace).

Since you are using the plasmid to make the standard curve, you would use the bp of the plasmid.

Double-check your math before you start, be very careful with your measurements, & mix thoroughly for each dilution.

I'd also suggest that you aliquot the standards into single-use batches & freeze them all. That way you can avoid issues from degrading your standards with multiple-freeze/thaw steps.

Good luck!

Yes! Real-time PCR, a hybridisation-based method, has become widely used for mutation detection. The systems are flexible with a number of different probe systems that can be used and there is additional flexibility in the design of the probes. Saima Qayum

Try Correlation matrix on R Programming. Try corrplot( ) package in R

Thanks a lot for your answer and suggestion professor Iman Hassan Ibrahim and Dr Abhijeet Singh

If you have amplification in qPCR, with using the qPCR product in gel electrophoresis, you must get corresponding band. If you are not getting it, there is something wrong in your electrophoresis protocol. I don't think primers are a problem in this case.

Hi Erina Noé Seiler, as Salma Aktar said, you can use β-actin or GAPDH as IPC to control inhibitions. You will find PCR sets for both in the following paper: https://gene-quantification.de/overbergh-1999.pdf

We use Qiagen extraction nuclei acid kit.@

Yusran Ady Fitrah

Hi Yusran, first of all, you need to make sure you have a correct and stable normalizer for the miRNA data.

for combination ROC curve, you need to use logistic regression model and picking a correct youden index later according to the sensitivity and specificity value, as a cutoff for your result.

hope it helps!

Dear Muhammad Haddad how are you doing?

Primer dimer is most likely related to the primers instead your lower expression. Check the complementary of each primer pair. In addition, the melt temperature is also important in this case. Each primer pair should be design in the same TM temperature. If you are using two-step qPCR reaction, the annealing and amplification occurs at the same temperature for standard 60°C. check your reagents data sheets.

Also, you can perform a conventional PCR reaction and run an agarose gel to access size of the band of primer dimer and your product.

In addition, there is a chance that could be genomic DNA contamination in your cDNA sample. In qRT-PCR analysis, your primers should be design in exon-exon junction or in different exons flanking a big intron. This avoids genomic DNA amplification.

Good luck in your research.

But, the 2009 procedure is very precise than 2002. We should not sacrifice accuracy for simplicity.

I am experiencing the same problem. I think it is not contamination but something with the oligo design.

Hi,

I have attached an article in which the protocol explains the use of conventional PCR for the detection of SARS-CoV-2.