Quantitative RT-PCR

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Questions related to Quantitative RT-PCR

Does anyone know the neutrophil marker for qPCR in human gut (duodenum, ileum, colon) biopsies?

Hello everyone. I have collected intestinal biopsies from stem cell transplanted patients in RNAlater and wish to quantify neutrophils only via qPCR. As we know that the mucosa of gut is filled...

26 November 2020 2,842 3 View

Can anyone help me troubleshoot QPCR for AAV titering?

So the lab that I work in generates a lot of AAVs and we titer them via QPCR. However, I've been noticing that the entire time, the efficiency has always been very low (anywhere from 40-80%) with...

23 November 2020 4,711 3 View

Using Circwave or JTK_cycle for qPCR data?

Hello everyone, My experiment is to assess the expression levels in cultured cells after the clock has been reset, or in mouse liver by qPCR. I have multiple timepoints and have performed...

22 November 2020 952 2 View

Data exclusion in qPCR 2^-∆∆Ct method?

I used triplicate for a sample in qPCR, and the Ct values are 26.0, 26.02 and 31.1 It is obvious that 31 is an outlier which may be resulted by technical error and I want to exclude it in the...

18 November 2020 4,554 3 View

What's the most optimal design for a qPCR experiment with 10 samples and 16 genes?

Hi! I have to perform a qPCR experiment in which I would like to evaluate the expression of 13 genes (plus 3 HKG) on 10 samples. I must carry the runs on a 96-well qPCR plate since I need to...

15 November 2020 5,630 4 View

Absolute Quantification: Serial dilution resulted in same CT values?

Hello, we are currently working on absolute quantification with SYBR Green qPCR. We excised from a plasmid containing the cDNA, a fragment, which will be amplified by the primers we used in our...

11 November 2020 940 4 View

Can I build a protein model which is not in protein data bank?

I want to use molecular docking to find the interaction of amino acids and solute carrier family 7 member 3 (CAT3), but the PDB of CAT3 is not available in the protein data bank or any other...

02 November 2020 3,438 12 View

QPCR considerations in analyzing samples from the field?

I am attempting to plan a qPCR experiment in which I evaluate the expression of multiple genes in various brain regions of fetuses, as a function of location in utero (intrauterine position,...

01 November 2020 5,892 1 View

Can anyone suggest suitable RNA immunoprecipitation (RIP) protocol from cell?

I would like to see RNA-protein interaction following transcription process inside the cell. I am wondering if there is any protocol similar to protein co-immunoprecipitation,which can bind the...

28 October 2020 3,125 3 View

Why do I see variation in melt peak across technical triplicates?

While reviewing my qPCR, I noticed that I had different melt peaks for samples run in triplicate. Is this something I should be worried about, or is this normal? I've uploaded a word doc showing...

27 October 2020 7,430 2 View

What is the best primer-design database and how to choose the best pair of primers i.e. what are the optimal characteristics?

Characteristics: product length, GC content, base repeats, melting temperature, exon-exon junction vs including an intron, self and 3' complementarity, the specificity of the primers, primer...

15 October 2020 944 7 View

How to calculate CT value with house keeping and GOI, without having any control for comparison? Is there any way other than absolute quantification?

Hi all, I have done qPCR with house keeping gene and GOI. My goal here is to check the expression of a particular gene and not to compare them with treated or untreated control. Is there a way...

13 October 2020 2,757 3 View

T Cell Isolation for RT-PCR?

I need a cell that will be HLA-DR positive on RT-PCR. For this, would it work if I directly isolate RNA from PBMC cells with Ficoll? Or should I necessarily culture these PBMCs and isolate T cells...

13 October 2020 3,678 6 View

What's the best multiplex RT-PCR detection system to buy?

Is someone working with the CFX96Touch Real-TimePCR System (Biorad)? How is your experience with this system? It is our first option for multiplexing qPCR systems because it is in our budget range...

28 September 2020 5,670 4 View

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

I dont know why im getting this kind of amplification curves. Anyone has an opinion respect to this issue ?

28 September 2020 8,481 5 View

Does polysome profiling in 4mL tube instead of 12 mL needs a scale down in ultracentrifugation time ?

Hello everyone, I recently started several polysome profiling experiment and something bugs me out. Every protocols I found use 12mL tube. They load their lysate containing ribosomes attached to...

20 September 2020 8,839 10 View

Early amplification in both NTC and NEC in qPCR (using taqman probes)?

Hi! I've been trying to decipher what happened to some of our runs with early amplification in the NTC and NEC. This has happened to us twice now. For background, we are using a mastermix of PCR...

10 September 2020 4,751 5 View

What happened to my fixed cell sample for mitochondria imaging?

I recently prepared some fixed cell samples for imaging. I need to observe the mitochondrial morphology. I overexpressed a GFP tagged protein, which localizes in the matrix of mitochondria. As is...

09 September 2020 7,914 6 View

Can anyone tell me the optimal conditions for N9 microglia cell culture?

I want to use N9 microglial cell line to evaluate neuroinflammation. As far as I know, different research papers report use of different culture medium (RPMI, DMEM, etc) with different...

07 September 2020 7,393 0 View

Can DNA copy number be quantified by normalizing to the total DNA in the sample rather than a reference gene?

I think I misunderstand the fundamentals regarding quantifying DNA copy number: When quantifying DNA copy numbers using droplet digital PCR (or qPCR) why don't researchers normalize to total DNA...

30 August 2020 1,619 3 View

Methods for detecting histone methylation marks?

Hi everyone! I'm trying to detect a specific histone mark (H3K9me2) by WB analysis and I´m not getting consistent results. I have also tried to use immunofluorescence and I was able to see...

29 August 2020 3,110 2 View

Different Ct values from repeat standard curve preparations and re-using same primers for different DNA containing same target region for qPCR?

Hello I've performed qPCR experiments for many years but I could not really figure out the following. First question: I've prepared standard curves from a DNA template (concentration determined...

21 August 2020 8,921 4 View

How to calculate viral titer based on qPCR measurement?

Hi there, I am having trouble calculating the viral titer based on a given copy number from a qPCR measurement. I have isolated the DNA from a cell pelett (5E6 cells) through a commercial...

20 August 2020 3,941 3 View

Can any one suggest me the best protocol for RNA binding protein immuno-precipitation for mRNA of interest?

I want to identify the unknown RNA binding protein(RBP) in few mRNAs of interest. I am looking for a reliable immune-precipitation method (or any other possible method) to find the unknown RBP...

18 August 2020 1,820 4 View

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