Question

Asked 28th Sep, 2016

Georgia State University

How to design qPCR primers for a 75 bp synthetic ssDNA?

I am a synthetic/medicinal chemist. We are currently doing a DNA encoded library of small molecules, we plan to label each small molecule with about 40 bp ssDNA. Now I can add few bp for primers in each side as the compliment for two primers. With this understanding I ordered a DNA of 80 bp with 20 primer binding site at each side. We did the PCR first and saw products only at 51-52 0C and then did qPCR with the same condition, ie; 52 0C. Unfortunately we only got the Ct values in range of 30-35 and not determined in low concentration. Reactions concentration was serially diluted from 500 uM ssDNA template.

Now the questions are,

Is the Tm value 52 is okay or we need to redesign the primers to make that in range of 55-65 ?

What other factors may be used to better the Ct values ?

Thanks every one

Georgia State University

Thank you Jose, We did not do the melting curve analysis. We will do that and see what is happening. Also the DNA template concentration we used were from 500 uM to 50, 5 and 0.5 uM. We got a Ct of 30 when we used 500 uM. I was skeptical about the Tm 52 and did not do anymore experiments till. But sure I will do some tomorrow and will discuss the outcome then

Cite

Paul Rutland

University College London

Bakhtiar Bukari

Deakin University

Michael J. Benedik

Hamad bin Khalifa University

Danung Nur Adli

Brawijaya University

Yuan-Yeu Yau

What is the difference between two base two logarithmic scale data points?

Question

3 answers

Asked 20th Feb, 2017

Alexandru Burcea

Hello,

I am analyzing a series of log transformed data points and I was wandering on the logarithmic scale how big is one point from the other?

The situation is like this: it's qPCR data and on one end I have the log transformed data point of 0.007 (2^(-dCq)) and on the other end I have 0.25 (2^(-dCq)). The 0.25 data point is clearly bigger that the 0.007 data point but by how much?

On the log scale it kinda appears that it's by almost 0.5 bigger, but if you divide 0.25 by 0.007 it shows that is 35 times bigger.

So what am I doing wrong?

(I do not have (2^(-ddCq)) because I am trying to observe the difference between controls and not between the treated groups so that I see if there is a statistically significant difference between the controls)

How to deal with non-detects in CT data when performing Delta Delta CT Method?

Question

3 answers

Asked 20th Aug, 2019

Tobias Haar

Input:

- RT QPCR dataset row CT values interpolate corrected

- 50 target genes

- 10 reference genes

- 3 different treatments with 3 replicates each

- 1 control with 3 replicates

Objective:

- Identify differentially expressed genes between each treatment and the control.

Problem:

- Many non-detects in reference and target genes.

Question:

1. Consider only reference genes for Delta Delta CT Method with no non-detects or how to account for missing CT values?

2. How to account for non-detects in the target genes without introducing bias?

3. For multiple reference genes, should the mean of all reference genes be considered as one gene when using Delta Delta CT, or should the genes be considered as multiple single genes?

What do you typically use as a standard for a qPCR standard curve and how do you calculate its mass/number of copies?

Question

11 answers

Asked 7th Nov, 2014

Ria Yagnik

I have done relative qPCR often before, but this is the first time I am trying an absolute standard curve. I've read several manuals/protocols, but they all seem to skim over a lot of the important details. Any detailed descriptions of how to set up an absolute curve would be highly appreciated.

First of all, I have read that many people use a single gene copied into a plasmid to ensure you know how many copies of your standard are in each dilution. Does it matter what gene/plasmid backbone, as long I know the size of both plasmid and insert?

Also, most papers/worksheets I've read say that the standard has to be "quantified by outside measures" before the dilution step, but don't explain what those outside measures are. Assuming you are using a plasmid at a certain concentration, what calculations do you use to go from that concentration reading to the weight or number of copies of a gene in an aliquot?

I'd appreciate anyone who has more experience in absolute standard curves who can answer these questions or a link to a manual which explains all the calculations/analyses needed - I have yet to find one.

Thank you in advance for your help

Can you help me with the Roche system for qPCR (DNA copy)?

Question

1 answer

Asked 23rd Mar, 2015

Olga Kgasha

The question is when I get a result on the Roche system for qPCR (DNA copy) is that equivalent to one bacterial cell and has the number of genomic units for that particular sequence been adjusted for automatically or does one still have to adjust for the number of genomic units.

For example: If I get 300 DNA copies of the IS6110 is that equivalent to 300 TB bacilli in the sample or do I have to divide this number by say 3 if there are 3 genomic units for the IS6110

What Ct value is good in Taq Man probe based q PCR to make a cut off value to determine the positive or negative status of the samples?

Question

11 answers

Asked 15th Apr, 2021

Tharaka Liyanage

Hello everyone, I am very new to q PCR and I am running a Taq Man probe-based q PCR assay to detect Toxoplasma gondii DNA in tissue samples of animals. I have generated a standard curve using 8 serial dilutions (starting from 500pg/ul to 0.00005pg/ul) using T. gondii g Blocks (synthetic DNA). My product size is 81bp.

At the highest dilution (0.00005pg/ul) I received a mean Ct value of 31.49. Do I need to use few more dilutions to get a higher Ct value? Is there any recommended reliable range of Ct values? The Ct value I am gaining from my standard curve will be used to determine whether the tissue sample is positive or negative for T. gondii. Any comments on the standard curve are also appreciated. Would love to hear the opinion of others who are doing q PCR.!

Does qRT-PCR amplify ONLY from fully spliced mRNA? Or could it amplify some pre-mRNA that still contains introns?

Question

10 answers

Asked 30th Sep, 2015

Brad Huffington

Does qRT-PCR amplify ONLY from fully spliced mRNA? Or could it amplify some pre-mRNA that still contains introns? I’m not a qRT-PCR expert, and before I reproduce a method from a paper, I want to be sure it is properly designed.

The scenario: A gene that is expressed in two forms – a full-length form, and a shortened form by activation of an alternate transcriptional start site. The alternate start site is in the first intron of the gene. Therefore the shortened protein is lacking the first exon, but otherwise identical to the full-length isoform.

The method quantifies the expression of each type using qRT-PCR (reverse transcription, then real-time PCR). There are 3 primers:

1. Forward, for full-length, within the first exon.

2. Forward, for shortened, within the first intron.

3. Reverse, for both isoforms, within the second exon.

-Full-length should be amplified by primers 1 & 3, because the first intron containing primer 2 has been spliced out of the mRNA.

-The shortened version will be amplified by primers 2 & 3, because the mRNA only starts at the 5’-untranslated region in the first intron which contains primer 2.

But, if some unspliced RNA is produced by the RT reaction, then primer 2 could bind to the unspliced intron 1 in transcripts of the full-length gene, falsely inflating the reported amount of the shortened variant. Is this a valid concern?

Random vs. oligo(dT) primers for reverse transcription: oligo(dT) primers should (I think) prevent or limit the amount of unspliced pre-mRNA, but will they reliably amplify the approximately 1 kb of transcript, from 3’-end to the 5’ first and second exons for which the qRT-PCR primers are targeted? Alternatively, can I trust random primers to reliably amplify only fully-spliced mRNA?

Contaminating genomic DNA: Can I be confident that DNAse will completely eliminate any genomic DNA? Because primers 2 and 3 will happily amplify directly from the genomic template.

I've looked for information on this subject, but I'm still not confident in my understanding. So any help would definitely be appreciated.

How can I analyse this absolute quant qPCR standard curve?

Question

9 answers

Asked 15th Oct, 2015

Lee Lee

I have been troubleshooting the qPCR standard curve for weeks.

A serial of 10-fold dilutions of porcine gDNA ( 10 ² - 10^-4ng) were tested using LightCycler 480. The run consist of 40 cycles of amplification. This qPCR assay were performed under same protocol and conditions for a year and it proved to be feasible. The result always give nice amplification plot with all S-curve were evenly spaced. And the amplification efficiency and r²value met the requirement in MIQE guideline too (result attached as AP.bmp)

Things getting wrong when I change to a new sample. Genomic DNA extracted from pork meat using Machery-Nagel Tissue DNA isolation Kit. I freshly diluted the gDNA into Sigma PCR-grade water as usual but the result turned up to be very wrong. The amplification curve getting far apart as the sample diluted more. I am lost :( (result attached as B.bmp)

Please share with me if you have any thought, or you know what happen to my serial dilutions assay. Your help is truly appreciated :)

QPCR statistical significance test?

Question

9 answers

Asked 23rd Mar, 2020

Angela Andrews

I conducted qPCR to analyse the gene expression of 5 genes. I used GAPDH as the reference/control gene. Everything was run in triplicate.

I have calculated the delta-Ct, delta-delta-Ct values and fold-changes (and log-fold changes) for each gene. I am wondering what is the correct way to determine significance in gene expression changes between treated samples and controls?

I have been using Excel (but also have access to Prism). I did a two-tailed unpaired t-test on the both thendelta-delta Ct values and the fold-changes, but I get different results in each. More results are significant when comparing fold-changes rather than delta-deltaCt. Which is the correct one to use? Any other suggestions?

Also, I would like to present my results as a bar chart of fold-changes. What should I use for error bars? The standard error (SEM)?

How to calculate proviral load in a qPCR using a house-keeping gene?

Question

1 answer

Asked 1st Jul, 2020

Sulav Shrestha

I am interested in calculating the proviral load of a retrovirus from the host genomic DNA using a house-keeping gene. What would be the best way for quantification without using a standard DNA template concentration?

Figure S6

Data

Jul 2012

Ontogeny of pri-let-7c-1 expression in pituitary development. Reverse transcription (RT) followed by quantitative PCR to analyze by quantitative RT-PCR to analyze pri-let-7c-1 expression in pituitary at E12.5, E15.5 and E17.5. The data were normalized by β2-MG mRNA. All data are presented as mean and s.d. (n = 4). (TIF)

Detection of cultured grouper viral nervous necrosis by real-time fluorescence quantitative RT-PCR

Article

Apr 2006

The probe and primers were designed by the way of TaqMan, and the method to examine the nervous necrosis virus (NNV) in reared Epinephelus lanceolatus by the real-time fluorescence quantitative PCR was developed. The main factors of the different concentrations of Mg2+ and the anneal temperature affecting the RT-PCR reaction were tested, and it sho...

Research advances of aptamers selection for small molecule targets

Article

Apr 2016

Aptamers are ribonucleic acid (RNA) or single-stranded deoxyribonucleic acid (ssDNA) selected by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers can identify small molecules, proteins, cells, microorganisms and other targets with high affinity and specificity, and have been widely applied in biology, medicine, food and e...

Got a technical question?

Get high-quality answers from experts.

Ask a question

How to design qPCR primers for a 75 bp synthetic ssDNA?

All Answers (2)

Top contributors to discussions in this field

Similar questions and discussions

Related Publications