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Questions related to Quantitative RT-PCR
Hello scientists! I'm working on gene expression stuff in rice plant, and I'm facing a little problem, which is that the starting quantity ,and expression of course,is so low on the qPCR n all...
16 March 2018 4,551 3 View
Hello everyone, I am working with qPCR and relative gene expression study of some candidate miRNAs. How many methods are there which can be used to normalize the PCR data? I know most famous is...
13 March 2018 9,703 4 View
Can someone give me an easy example of how to normalize qPCR data against spike-in control? Thanks
06 March 2018 9,654 1 View
I am trying to build up 3 standard curves for 3 sets of primers for different reference genes, in order to later use one with the best efficiency. (for SYBR green qPCR, gene copies number...
28 February 2018 8,394 3 View
We are looking into liquid handling systems for pipetting qPCR plates (384 well) and for NGS library preps (amplicon seq). There are many machines on the market that are able to do that (Biomek...
26 February 2018 8,814 5 View
Hi there, I am currently reading a document from applied biosystems called "Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR"....
23 February 2018 9,001 5 View
I am working on neural differentiation of dental pulp stem cells (DPSC). I am checking some ion channels that play role in the differentiation and want to do an absolute quantification of my qPCR...
12 February 2018 1,853 3 View
Do I understand right: when running qPCR with SYBR green detection, it is important that two amplicons (GOI and reference gene) should be of the same lenght ? Once It is supposed to be relative...
08 February 2018 4,779 5 View
Hi. I'm having a problem in analyzing my qpcr data. So now I had calculated both the ddCt and 2^-ddCt. And as far as I know, for ddCt method, if the experimental group shows a negative value, then...
05 February 2018 4,376 1 View
Hi fellow researchers, Which house-keeping gene should I use as reference for normalization of candidate Long non-coding RNAs (LncRNA) in hypoxia induced samples. From literature the use of GAPDH...
27 December 2017 3,375 3 View
Dear all, I want to measure the expression of 9 different genes all belonging to the same gene family. I want to see which of these 9 genes comes to expression in the bean of Phaseolus...
07 December 2017 8,630 3 View
I want to calculate a gene expression rate of 1 gene of interest and 2 reference genes (GAPDH, 18S) over different timepoints and accounted for primer efficiency. Since im working with RNA...
07 December 2017 6,347 1 View
Hi, I am currently trying to evaluate multiple qPCR plates in the Biorad CFX software. I know that you use the Gene Study option for this, however I'm afraid that it's not working the way I need...
29 November 2017 3,000 6 View
I am trying to define a standard curve for the primers that I have designed for qPCR and I ordered to sythetize the correpondent oligo for test the primers in different concentrations, but I don't...
29 November 2017 5,332 2 View
1) I have to use "Log2FC" (which is basically negative ddCT values) and "bar-graph*" to show my RT-qPCR data. Is it more appropriate to use “confidence interval” or SEM as error bar? (*I agree...
26 November 2017 8,626 11 View
I'm doing phagocytosis assay of bacteria in whole blood with FACS. Since using the confocal microscope, I've seen some bacteria attach to the surface of the cells I've considered using of Trypan...
17 November 2017 4,942 8 View
Hello dear collegues. Im using sybr green 1 qpcr assay for evaluation of relative mitochondrial copy numbers in dna samples from human blood. Im using primers for nuclear and mitochondrial genome...
11 November 2017 649 4 View
I mean, a possible mechanism of action?
03 November 2017 9,519 4 View
I am culturing bEnd.3 cells (mouse brain endothelial cells). I have noticed my Ct values for the same cDNA input amount increase. For example, GAPDH went from Ct 13 to 20 when using cells from...
01 November 2017 6,255 3 View
Any explanative thoughts or information? Will the 2nd or 3rd melt of products attribute to some improvement for their detection?
21 October 2017 8,656 3 View
Hi, I performed a qPCR assay, using the StepOnePlus qPCR instrument (Applied Biosystems) and Qiagen qPCR kit (SYBR Green kit). I chose the fast ramp speed setting (as recommended by the kit). I...
13 October 2017 9,409 3 View
Hello colleagues, I have designed some TaqMan probes for using in ddPCR, and to save expensive ddPCR reagents I decided to test the probes in conventional RT-PCR machine first. The probes are...
11 October 2017 8,537 5 View
Dear All, I am working on determination reference gene for qPCR, I think normfinder is quiet simple software to assay our best reff gene. Whereas we couldnt just simple input our Ct value, but we...
29 September 2017 1,411 0 View
I've used a SpeedVac for concentrating pDNA after extraction with a commercial kit and lost a lot of material. While the volume was reduced to around 20%, the increase in concentration was very...
25 September 2017 7,488 3 View