Baselines in Real-Time PCR -- Ask TaqMan®: Ep. 5
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- Опубликовано: 9 сен 2012
- Submit your Real-Time PCR questions and watch the rest of our videos at ow.ly/bQh0l. Life Technologies Sr. Field Application Specialist Doug Rains helps with the understanding of baselines in Real-Time PCR. We're looking at a fairly standard real-time amplification plot. We have some nice curves, each of which has the familiar geometric phase, linear phase, and plateau phase. So far, so good. But what's all this . . . junk in the early cycles? Well, friends, if you said "junk," you were right. That's right, I said it -- junk, trash, waste, detritus, garbage, otherwise known as noise.
It's the stuff we see before our actual signal from amplification gets high enough to overcome that noise. And, as the rather impolite adjectives I used a second ago would suggest, it's completely useless to us. This noise does have an effect on our curves. Our job is to minimize that effect by effectively subtracting out the noise. We do that by establishing what's known as a baseline -- a cycle-to-cycle range over which only noise can be seen, prior to the appearance of curves. Once established, the software will effectively subtract out the noise on a well-by-well basis, greatly improving the quality of our data.
Let's switch the Y-axis to linear scale for a moment to illustrate the effect of baseline subtraction. Here's our data prior to baselining. Note how every sample begins from a slightly different spot on the Y-axis, causing our geometric phase data- this curvy part over here when we're in a linear scale- to look horrible. But once we subtract noise, every sample begins from the same point 0. And as a result, the data clean up nicely.
The value we get after normalizing for background noise is something called delta-Rn. If you ever look closely at a log-scale amplification curve- the one we're used to seeing- you'll notice that delta-Rn is what's graphed on the Y-axis.
But before you go, just note that there are two ways to set baselines in Applied Biosystems® real-time PCR software: manually, and automatically. If you do it the manual way, you set the baseline range under Analysis Settings. You either set it for a single assay, in which case all wells for that assay get the same subtraction . . . or you can go under Advanced Settings and set wells individually.
Better yet, just use the default setting of Auto Baselining. With this selected, the software figures out how much noise needs to be subtracted from each well individually, and, as such, generally produces the best results.
So why have a manual feature? Well, Auto does fail on occasion, especially with some SYBR® Assays and non-standard chemistries. You'll know auto has malfunctioned by the shapes of your curves. If they look more S-shaped than they should, it could be that auto has misapplied the baseline and set the End cycle too low. As a result, not enough noise is being subtracted, and the curves take on a strange shape. To fix the problem, switch over to manual mode for that assay and raise the End cycle until the curves take on a regular shape.
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A video like this is worth 10 pages of heavy documentation, which is often incomplete and inconsistent. Priceless. Want more!
If a picture is worth a thousand words, a video is worth a million. Thanks for these!
Thank you, TaqMan®! You saved me from repeating my qPCR _and_ thought me some fundamentals in your software. I watched other videos of your previously. Love your work!
thank you sooo much for the simple explanation
i love your attitude
thank you :)
Yes queen !!! weeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeerk ! love it
Very clear explanation, thank you.
Our pleasure! Thank you for giving it a watch!
Hello,
I am struggling with the Ct values I got from my lab.
For my gene of interest I got no amplification for the negative control cells, for the test cells I got 30.935 and control cells 33.759. For my reference gene I got 30.310 for negative control, 28.260 for test and 28.022 for control. I got wrong results since the negative control where there should not be amplification and the ct values are too high. However I need to calculate the delta ct for test (I got 2.67) and for control (I got 5.7) to compare house keeping and gene of interest. But what do these values really tell me? And for the double delta Ct I got 8. What does it mean?
I hope to have a reply from you soon!!
Best wishes,
Natalie
taqyou very much.
It's no P(CR)oblem at all!
Yours was better. Respect.
How to recover the data if you forget to save after run
This video is incredibly helpful. Best one 👍
We're happy to help!
@@thermofisher what is meant yellow triangle flags with numeric 2 in the well plate after competition of result?
@@thermofisher I got many yellow flags in my results with no determination of quantity, yet there was definitely DNA there.
Can you help ?
@@iqrach.9773The triangle flags are QC Flags, details can be found in QC summary(in the Software interface).
If you are doing a AQ experment without any quantity in the unkown sample, you may check the Standard Curve first to see if the standards are properly defined.
You may send the data file to TechSupport team to get more help.
I am setting baselines to normalize my Ct values. Should I manually choose an end cycle where all of the plots for my target converge or relatively at the same point?
Thanks for your reply. Lifetech emailed me yesterday and I can now download the program.
I used SYBR-Green with L29 & L15 reference ribosome-genes to identify Vibrio bacteria from biofilm samples. The result I'm trying to get out of it is the number bact/sample & the original number of CFU (Colony forming units) per area of biofilm. So how do I go about interpreting the results that the qPCR 7500 model gives me to answer these questions? (A collegue is working on FISH results to corroborate).
Is there a video that explains the data that I get in an exported xls-file? Because I don't have the qPCR software on my own computer, this is all the data I've got. I don't have the serial number of the machine I used because I don't have access to it any more, but have emailed the company for an alternative download. No reply yet though (~1 week ago). Thanks for great videos!
10/10 would recommend
Numbers don't lie! Thank you for the support Chaenne.
Sorry, I don't have the software version but it was running on WinXP.
Hi ...i need to check the expression of some genes can you do a tutorial on how to assign samples qnd target genes? it is really confusing especially when you have triplcate for each sample..
amen
goo day! this episode is helpful.. however, i have some concerns.. i did qpcr to determine the gene expression of cytokines in different treatment groups... when i ran my samples, most of it are flagged saying exponential algorithm failed. when i look at the amplification curve, its unacceptable... shall i discard my experiment? and repeat the process?
***** the problem is, even my positive controls are flagged, no amplification... i could not determine the possible reason. :(
+KC Doysabas Hello! If there is no amplification, even from the positive controls, I would check the samples and assays. How much sample are you using? Have you tried a dilution series, say from 0.1 to 100 ng of cDNA? What type of assay are you using? Is this TaqMan or SYBR? Is the design good? Have they worked before?
I am attempting to determine a male plant from female plant by locating the Y-chromosome in a qPCR Machine. Right now I am still searching for the knowledge to do so. My question is, do you think this will be possible with a PCR machine? And have you ever done something like this?
Thankss!
+Jake Richard This should be possible. However, you would have to determine the appropriate target sequence for whatever plant species you are working with. Here is a reference that did something similar:onlinelibrary.wiley.com/doi/10.1111/j.1755-0998.2008.02344.x/abstract
Hey TaqMan, how can I visualize my (running) experiment in QuantStudio , if I have closed the software by mistake? Thanks
Hi Rafi. Thanks for your question.
Unfortunately, if you have closed the software on accident, you’ll have to wait until the run is completed before viewing the data in the software. You can still view the experiment in real time on the instrument itself if you have accidentally closed the software though.
For additional technical support, please contact us at thermofisher.com/askaquestion. Thank you!
Hi
1. Iam Using SSIV for cDNA preparation. What is the max conc of RNA that can be used in a 20uL reaction setup when using SSIV?
2. What is the maximum vol/Conc of cDNA that can be used in a 10uL qPCR reation?
3. Iam doing relative expression studies for some cytokines and could able to see the flags reporting exponential algorithm failure. How to resolve this problem?
Need some help.....
+Ratnakar Reddy The maximum RNA input for a 20 uL reaction is 5 ug of total RNA. In a 10uL qPCR, we would recommend that the undiluted cDNA not exceed more than 10% of the total reaction volume (ie, maximum of 1 uL of undiluted cDNA in a 10 uL qPCR reaction). Those flags typically indicate that there was no amplification in the well. I would review the curves and see if they look normal or not. If there was no amplification, I would check the sample - in some cases you may not expect amplification (low or no expression) from a particular sample, and so such a flag would be expected and can be ignored. Thanks for asking, and let us know if we can help with anything else.
do you have a software that is compatible with macbook?
+KC Doysabas The Applied Biosystems Analysis qPCR Analysis Modules allow for data analysis that is PC independent. Simply access the system from any computer (PC or Mac) using your browser. You can find out more here: www.thermofisher.com/us/en/home/cloud/all-analysis-modules.html?icid=cloud2#qpcr Hope that helps.
Why there is exact count of cycles in every test needed
Hi,
In my standard curve experiment, in the graph of delta Rn vs # of cycles, I don't find the plateau phase. even if I have put 45 cycles, the run completes without reaching the plateau phase. I see only linear and geometric phase. Please suggest me what I can do and what is the possible reason for this.
Hello! First, thanks for watching our video. Second, your description sounds like the target is expressed at very low levels, in which case you may not see much of a plateau. Have you tried using some sort of positive control which would have robust expression? Another possibility is that there are inhibitors in the sample which is hindering the amplification. If you want to post more details and/or data in our community here (abcommunity.lifetechnologies.com/welcome), and we can examine the issue further if you would like. Hope that helps, and let us know if you have any further questions.
Thank you..but if I increase the # of cycles can I reach my plateau phase??
We would not recommend to go past 50 cycles. The Taq enzyme activity will be reduced at this point in the reaction. If the signal is still low this means the target is very low abundant (single copy range) and you are at the limit of detection. Any data at this point is likely not quantitative.
Thanks..:)
Hi, please some one helping me, I already extracted RNA from the colorectal rat tissue then CDNA, the concentration of my RNA was 15.12 ug/ ul. Then I want to express PPAR genes, but I did got any Cq value for target gene just I have the Cq value for my keeping house gene. The concentration of rna in CDNA also high. Please help
Hey Layla,
If you can fill out the form below with some details on your issue and how to reach you, someone will follow up with you shortly:
www.thermofisher.com/askaquestion
First you need to verify that is the PPAR exprssed in your sample.
Then check the PPAR assay you used, if the primers are well-designded?
Can you explain about undetermine ct ?
Thank you for your question. An undetermined CT is usually indicative of no amplification. The software is designed to set a default threshold 10 standard deviations above mean fluorescence generated during baseline cycles. If the target does not amplify, it will never cross that threshold line and therefore, you will see an "Undetermined" in the CT value.
For additional technical questions, please feel free to contact us as thermofisher.com/askaquestion. Thank you!
How much dna quantity required for sequencing after RT PCR
Hi Kiran. The amount of DNA required for sequencing would depend on the sequencing platform.
If you are using one of our sequencing platforms, please contact us at thermofisher.com/askaquestion so our sequencing technical support team can assist you.
If you are using another brand’s sequencers, please contact them.
Thank you!
This is the holocost isn't it
You should use a mic. Plzzz
I want to know if my DNA forensic manipulation and torture experimentation is taking place
Liars