The Significance of Early Ct’s - Ask TaqMan #35

[@dr.vikrambhoomi-scientist]

Please post as take one sample as example and explain total analysis of RT-PCR

[@sofianbio4997]

Can u please explain that on the device from zero to last step and give us a calculator example as a mathematical way thanks

[@radjasalavatore7835]

Hello . Can we calculate viral load from rt-pcr (qualitative ) ?!

[@randomstufffromachristian2287]

This was awesome Thx, God bless ya

[@trungnguyenkhac4834]

Very good and funny videos bring a great sense of entertainment!

[@lientran-wx6rf]

the video image is too poor, you need to fix it more

[@pravakarsunuwar7680]

How to correctly set threshold value for reaction sets?? We put 100 for biorad cfx96. Please elaborate.

[@thermofisher]

Hi Pravakar, Thank you for your question. The threshold should be set during the exponential phase of the plot. As we are not familiar with the BioRad software, I would recommend contacting BioRad Tech Support on how to set the threshold in their software. If you have questions specifically about using our software, please contact us at thermofisher.com/askaquestion. Thank you!

[@jjwhyte14]

Nice, professional presentation. I'll be that guy who has to find one thing wrong, no matter how minor: At 1:44, the SI unit for nanogram should be lowercase (ng), not uppercase (NG) as is shown in the presentation. Otherwise, great stuff.

[@Danielle-hf7rm]

Can a CT value be too low? If so, what does it mean and what is the border value?

[@thermofisher]

Thank you for your question Danielle. When starting a qPCR experiment, it is good practice to determine a dynamic range to see what the qPCR efficiency is at more concentrated samples compared to less concentrated samples. This will also provide you with an idea of what CTs you are expected to see with the more concentrated samples. If you observe a low CT value and that is reliably replicated, then it could be considered valid. If you need additional information, please reach out to us at thermofisher.com/askaquestion. Thank you.

[@MrSAMSARA9991]

What do you think about Atila RT pcr instrument for Covid-19 detection? On wet samples?

[@thermofisher]

Thank for your question. Unfortunately, as we do not support the Atila Biosystems test, it would be good to reach out to them to determine whether wet samples could be used for COVID19 detection. If you have any specific questions about our instruments or our COVID19 TaqPath combo kit, please contact us at thermofisher.com/askaquestion. Thank you!

[@ashwatiravindran7929]

Can there be a cq value as low as 3? If it cannot be, is it because of the background noise?

[@thermofisher]

Thank you for your question, Ashwati. Low Cq values are not always accurate as it can be do to fluctuations in the fluorescence or an issue with mixing. If you would like our technical support team to take a look at your data, please reach out to us at thermofisher.com/askaquestion. Thank you.

[@muffinman1]

Informative and simple

[@stainlesley2ride]

I used double delta ct method to calculate the fold change. How can I plot a graph showing the difference between control and treated group owing that I have one fold change number(assuming 5.2) ? Thank you.

[@thermofisher]

Hello! The RQ of your control group would be 1, since RQ = 2^-ddCt, and ddCt for the control sample is zero. So you could plot as a bar graph of 1 for the control sample versus 5.2 for your treated sample. Let us know if you have any other questions.

[@stainlesley2ride]

Thank you for the quick response. Follow up questions. (1) Is it possible to show the error bar for control group? (2) Does a positive number from RQ = 2^-ddCt mean up or down regulations? I am seeing different things from different articles

[@livais272]

what does delta RN means??

[@thermofisher]

Hi Livais. Thanks for your question. Rn is the reporter (fluorescent signal) that is normalized to a reference dye. The deltaRn is the Rn minus the baseline. If you have any other technical questions, please reach out to us at thermofisher.com/askaquestion. Thank you.

[@nabeelazaman8927]

How dct value and RQ value are related? Is dct value is inversely proportional to the gene expresseion? If dct= target gene Ct - Ct of endogenous control

[@thermofisher]

Hi Nabeela. Thank you for your question. Your answer can be found in our PCR handbook on page 42. If you have any additional technical questions, please reach out to us at thermofisher.com/askaquestion. Thank you! www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/real-time-pcr-handbook.pdf

[@fatemahaalhashem2308]

Great explanation 👌 thank u so much 😀

[@hollyferguson9392]

are there any videos which could help with understanding efficiency data? I'm trying to decide which genes can be run in singleplex or duplex and getting very confused with what to do with my output!

[@thermofisher]

Hi Holly. This one is a good overview of efficiency and what it means: kzbin.info/www/bejne/n3vPmJ-Ie6ydptl9n addition, this application note gives a good outline of how to validate your duplex reactions: tools.thermofisher.com/content/sfs/brochures/cms_076529.pdf

[@shaikhamadsharif8893]

Can we make cutoff for Ct value in qualitative PCR

[@thermofisher]

Thank you for your question. Could you please contact our technical support team at thermofisher.com/askaquestion with additional details and we can further assist you? Thank you!

[@anitasharma-ef5ft]

What does high CT value represents

[@thermofisher]

Hi Anita. Thank you for your question. CTs are correlated to how much of your target you have in a particular sample. If your CT is high, this means that there is less of your target and it’s taking more cycles to reach the threshold resulting in a later CT value. If you have more of your target, it will take less cycles to reach the threshold resulting in an earlier CT value. For additional technical support, please contact us at thermofisher.com/askaquestion.

[@calarzt3453]

@@thermofisher if CT high in 45 cycling .we need more cycling to give a positive results in covid_19 Or end the run. In same sample or repeat the test in anthor sample ?????

[@yasirarfat8777]

At day 2, test was performed, ct value was 29 now after 35 day test was repeated ct value is 31. What may I conclude? Waiting for your kind guidance

[@jahirhasan1170]

@@thermofisher how this threshold line is plotted ?

[@milanshrestha7363]

what if ct value is 25 ??

[@thermofisher]

Hi Milan. Thank you for your question. A Ct of 25 is acceptable. If you have any additional questions, please reach out to us at thermofisher.com/askaquestion. Thanks!

[@agentgunnso]

thanks for your video!

[@thermofisher]

We're glad you like it! Thanks for watching.

[@DA-sj2gw]

If sample 1 contained 40 ng of DNA, how much DNA was there in sample 2? 2 fold dilution Cq = 1 Sample 1: cq = 20 Sample 2: cq = 22

[@thermofisher]

A two-fold dilution would give a 1 Ct difference. A 2 Ct difference would therefore be a four-fold dilution. (2^x = fold dilution, where x is the Ct change)

[@ShakespeareCafe]

Miss the Original Taq Man

[@nataniyaandjeremiahinwonde3770]

My ct value 11.9

[@pamelamariaa]

Very good. Helped me.

[@thermofisher]

Glad to be of service!

[@dr.atouisttx]

I shouldn't have drank before watching this...

[@Queenkz13]

He looks like a mike. This was a really helpful video though.

[@shreyashah9598]

Also he kind of looks like a Bob or Bill!

[@hadramcoltzau6135]

the golden pipette haha

[@thermofisher]

Can you believe Hollywood turned down our pitch for a "Scientist with the Golden Pipette" movie in the James Bond franchise?

[@hadramcoltzau6135]

@@thermofisher haha there must be something wrong with these Hollywood directors.