RT-qPCR troubleshooting. What could be generating this kind of result?

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What happens if i defrost the pcr buffer wrong? How is this evidenced in my pcr results?

I would like to know the consecuences of wrong reagent defrosting. does this affects reagent concentration? how do i evidence this fact on my pcr results? thank you very much for your help

26 January 2021 516 1 View

What is your perception on using visual analysis or quantitative approaches to evaluate data from single-case research designs (n of 1 trials)?

Results of single-case research designs (i.e., n-of-1 trials) are often evaluated by visually inspecting the time-series graph and computing quantitative indices. A question our research team is...

03 March 2021 687 1 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

How to quantitative analize the data of TNBS assay for gelatin?

Estemeed colleagues, I found some issues regarding the quantification of the data for TNBS assay. There are different protocols on how to perform that but it is clear to me the "fil rouge" that...

02 March 2021 2,616 1 View

Sample dilution for quantitative analysis using ELISA?

If the detection range is in ng/ml but the reference range is in ug/ml for a molecule or protein in serum or plasma .how to dilute and what is the initial volume to be taken for quantitative analysis

02 March 2021 7,670 3 View

Do you know good and user-friendly programs to measure plant quantitative traits?

For my research i will need to measure plant quantitative traits (especially leaves area and roots length, but would be nice to add some more information). I recently discovered...

01 March 2021 5,035 2 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View

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Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 View

Distribution of particles in metal matrix?

How would define the "good" and "poor" distribution of particles within the matrix. Is there a quantitative solution to assess the distribution of the particles, which then having denotation of...

28 February 2021 7,397 8 View

MiRNA qpcr problem?

Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.

28 February 2021 5,008 4 View

Navonil Gogoi

Christian Fernando Montoya Estupiñan By looking at your image are you using BioRad CFX96 PCR? PCR amplification is exponential in nature so you should see a sigmoidal curve not linear ones like yours. You can confirm by running your samples on a gel and check for PCR products. Try running a positive control. Start by making everything fresh make new working stock of your primers and probes, new nuclease free water, new master mix if possible, and add a positive control. May I know which fluorescent dyes you are using? Please also check your NTC.

Katie A Burnette

Looks like you had no measurable increase in template. You should have a sigmoid curve, not a gentle positive slope. What did your positive control look like? Given that you've only highlighted a few of your samples, I'm guessing that the rest looked different. In that case, check your technical replicates & housekeeping gene to verify that you have cDNA from those samples.

Sayanta Bera

Christian Fernando Montoya Estupiñan Please go for a melting curve analysis to make sure there are no misamplifications. It would be nice to do a primer efficiency test also, to me it seems like the primer efficiency is not good. Designing a new set of primers specific for your GOI should help.