Quantitative RT-PCR

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Questions related to Quantitative RT-PCR

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 962 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 2,786 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 3,701 6 View

Is there anyone who did the isolation of the apicoplast from Plasmodium falciparum?

I need a person/team familiar with the isolation of apicoplast from Plasmodium falciparum.

11 October 2020 1,513 3 View

Let me know how can I knockout a gene Insilico approach?

I've designed gRNA by using CRISPR-Cas9 protein on a computational based. I fail to understand now, how to delivered CRISPR-Cas9 with gRNA to the target region or to reach out to the results of...

11 October 2020 5,979 1 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 5,706 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 8,495 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 8,870 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 4,778 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 7,926 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 7,407 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 1,634 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 3,120 2 View

Methods for detecting a specific histone methylation mark (H3K9me2)?

I´m trying to detect a specific histone methylation mark (H3K9me2). Which is the best way to detect this type of modifications? I have tried WB analysis but I don´t have consistent results. I also...

29 August 2020 4,993 1 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 8,948 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 3,967 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 1,838 4 View

Can oligo hexamer primer produce sequencing error?

Hello everyone I conducted sequencing prosedure for RNA genome segment (about 700nt) , for this reason I employed the random hexamer primer for cDNA synthesis . after sequencing (by sanger...

17 August 2020 4,154 3 View

Can qPCR method validation be done using a conventional PCR machine and a plate reader?

I am working for a small CRO which hopes to validate my qPCR method prior to purchasing a qPCR machine. I have attempted to validate my method using a conventional PCR machine while measuring...

05 August 2020 4,095 4 View

Is it advisable to do LUMINEX assays in duplicate or triplicate?

We would like to do a LUMINEX assay with the Bio-Plex Pro™ Human Cytokine 27-plex kit.

26 July 2020 2,877 1 View

Difference between Caliberator and Normalizer?

Just wanna make sure i understand right.

25 July 2020 5,365 4 View

How to use ARG-Annot to screen for AMR?

I am currently screening for antibiotic resistance in strains of Staphylococcus aureus, the strains that I am using are already assembled, sequenced and annotated. I have used ResFinder to screen...

09 July 2020 5,538 2 View

What type of research should do when obtained zebrafish genes knock-out mutant?

I have got zebrafish knock-out mutants with crisper-cas9 method, I only observed mutant possessed obvious phenotype and did genes qRT-PCR analysis. however, I didn't know how to design next step...

28 June 2020 3,439 0 View

With a CMV promoter, does transcription always start at +1?

I know the theory says that transcription starts at +1. However, sometimes transcription can start at a different position (see for instance the article "RNA Polymerase II Activity of Type 3 Pol...

18 June 2020 5,790 1 View

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