What are the best tools for correction for efficiency and interplate calibration?

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

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...

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No title

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

01 March 2021 5,311 1 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Genome-wide gene identification protocol ?

Is there any book chapter/book, webpage or research article available to understand genome-wide gene identification?

28 February 2021 8,095 1 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

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

I have two groups of brain samples, control and treated for example. It was total RNA nova seq sequencing. I tried all the available pipeline like: star+rsem+deseq2, Hista+stringtie+cuffdiff,...

27 February 2021 356 6 View

Runs of nucleotides in Sanger sequencing?

I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...

27 February 2021 547 3 View

Fulvio Celsi Popular answer

use LinRegPcr http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR

free and easy to use...and you have the reaction efficiency and from this you can calculate DDCt....

Fulvio Celsi

Stefan Rödiger

A recent paper by Ruijter et al. 2012 discussed several important aspect. Definitely worth a read.

https://www.sciencedirect.com/science/article/pii/S1046202312002290

Johannes Denk

I can recommend GenEx software. The software allows to normalize interplate calibration with Interplate Calibrators. It also allows to correct for efficiency and provides a lot of useful tools for statistical analysis of your qPCR data.

It is easy to handle and raw data can easily be converted into the appropriate data format. It is not freeware but a 30-day trial is provided here:

http://www.biomcc.com/genex-contact-data.html

Check also the GenEx Help Guide:

http://www.multid.se/genex/onlinehelp.php

and the GenEx user guide (attachment) to make sure it is the right software you are looking for.

Claudia Mandolini

Use GenEx as suggested or internal calibrators that you have to put in each run, to see how ct value change from one run to another one.