I think qPCR is the best way for quantification os gene expression and it is also well known.

Definitely real time PCR!

It is more accurate and more sensitive than the branched DNA quantification.

Branched DNA quantification used to be a good method around the turn of the centurey for example in virology, it was used a lot (e.g. HIV RNA quantification). At present, most, if not all clinically validated platforms are based on real-time PCR as these are more sensitive and accurate.

The good news: You will also find it more easy to optimize a real-time PCR assay!

qPCR or digital PCR

qPCR definitive; I have expirience with both

i think its qPCR

Hi, Mochamed, the qPCR will give you relative mRNA level of your gene of interest to compare with marker gene. So, you results may largely dependent of the standard you use. Moreover, qPCR give you reliable data if you use cell culture, ea. all cell in the population have similar mRNA level. In the case you have a multi-cellular organism, you should be care about data interpretation. In this case some cell may increase mRNA level in response to stimuli or in the mutant, but in the same time some cell may decrease mRNA level in response to the same stimuli. This is highly dependent from cell fate/stage. Please, consider these points and try to isolate RNA from as much as possible homogenous cell type. Good luck!

I think that qPCR is one the best method for expression

real time PCR by sybr green (if you design the primer oligos) or Taqman (ready to use from commercial provider) would be the best

I think that qPCR is the best way for gene expression quantification.

Real time PCR is the golden standard. Depending on your assay you might find one to work better than the other. Personally I prefer qPCR, easier to design, if you are careful picking up your standards. Plus you can use SYBR green or probes, depending of your region of interest. Though, there are cases that bDNA assay has worked better (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2259472/).

I agree with Demosthenis, qPCR is a golden standard for quantification of mRNA level in the homogeneous cell population (cell culture, for example) when each cell have almost similar mRNA level of the gene of interest. However, when one want to investigate mRNA level in the muluticelluar organism, in which mRNA level were different among cells, the interpretation of the data is questionable. In this case one should do in situ.

qPCR using Taqman probes is more specific than SYBR green.

OK, RT-qPCR is very popular and "gold standard" assay for gene expression. However branched chain amplification is also very accurate when used within the assay limit of detection range. I use bDNA for multiplexing many targets-much more practical than qPCR. Also you can include more housekeeping genes that will really smooth out any gene expression bias. Lastly, as it was mentioned bDNA is hybridization based. qPCR is not infallible as it relies on enzymatic activity and amplification efficiency should be determined for any assay in order to be accurate. So PCR inhibition can be a problem in some samples and skew results. Bottom line: if you are using cell line RNA or fresh frozen RNA and only interested in a singleplex reaction, go with qPCR. But don't discount bDNA for multiplex gene expression-it works well!

In an internal study comparing a bDNA assay to an RT-PCR assay, my colleagues and I found that the assay-to-assay reliability (correlation of results from repeated assays of the same biological samples) ranged from 0.96 to 0.98 for the RT-PCR assay, but only 0.58 to 0.74 for the bDNA assay. Thus, RT-PCR is by far the more reliable (reproducible) method.

Real time PCR, but for gene expression its Real time RT-PCR.

To detect mRNA level, real-time RT-PCR is a very convenient assay. You can do absolute quantification using Taqman probes or relative quantification (compare with housekeeping gene) using SYBR green.

Thanks William Kaemmerer. Is this work published by any chance? or just for your own sake. Thanks for sharing this with us.

I agree with previous comments, Real time qPCR is a great assay!

Quantitative Real-Time PCR would be efficient and more reliable for gene expression, keeping in mind that the target gene should be known. You could use sybrgreen for fast and accurate results. Also house keeping gene (reference gene) will be the key of your relative expression. there are many softewears for such comparison.

qPCR was good last year, currently dPCR (digital) is new, but convenient and well established method.

While most answers above are indeed correct there is a problem with the linear range of both techniques. This issue is more pronounced with branched DNA. Also most labs have more experience with qPCR, thus that would be the safest bet.

Having said that, I agree with Vladimir: the only truly reliable quantification technique is digital PCR as that does provide with actual numbers while both qPCR and Branched DNA give only relative numbers (relative to standard).

Both are equally good for gene expression quantification. It depends on the type of data you are looking for your study. Since there is no mention of that in your question, it would be too premature to comment on your querry.

I would prefer qPCR with intron-exon junction primer.

Real time PCR. I never used branched DNA.

Thanks everyone for valuable comments. I would also personally prefer the real-time qPCR, although I have no experience with the bDNA approach. The qPCR is very convenient and straightforward. However, several factors have to be concerned including the specificity of oligos in particular. A keen qPCR assay will follow the predefined code and conducts by MIQE guide lines. You may go through the MIQE standards in this article: http://www.rdml.org/clinchem.2008.112797v1.pdf. The qPCR should be carefully conducted when you analyze couple of related genes such as isoforms with high sequence identity. Because, the corresponding oligos could be non-specific to our target genes. I guess, these facts will help you; and good luck!

http://www.rdml.org/clinchem.2008.112797v1.pdf.

I think that "Real time qPCR" is a best method for gene expression analysis.

I think the qpcr, by the method of comparative ct.

Real time PCR

If use relative quautification in real-time PCR method, it is importent to carefully choose housekeeping genes that should be expressed at relatively constent level under your experiment condition. Mean value of 2-3 housekeeping genes are needed for normalization.

My experience of 15 years with PCR advise to use with REAL TIME Amplification. As that is the target amplification as well as it tells you each information at each step and every cycle i.e, IC, No of viral or bacterial load, standards and amplification at each step.

RealTime (or RT-qPCR ) is quantitative and permit to look small difference between treatments, semi quantitative PCR isn't indicated because a lot of variable can alternate the final results (method of quantification, different quantity of stain in the gel, ecc...) so can give some summary information about the mRNA accumulation.

I think real time PCR is better and more accurate

RT qPCR is better.

Real time qPCR surely

You can check this

http://www.hivtestingconferencearchive.org/hivtesting2007/poster/poster19.pdf

and this

http://jcm.asm.org/content/44/3/729.full

Quick look,

qPCR is less labor and more sensitive for the specific mentioned diseases. G.luck

Thanks Hatem. Appreciate your input.

Generally speaking,if you try to exame some genes expression which you know are reraly and lower expression , like transcriptoms, you would choose Real time PCR because it is a more sensitive and accuate approch. if you just want to measure the gene expression while not interfering its performace, like in viral RNA or in plasmids, then BDNA menthod.

Real time PCR

Real time PCR

I am confident enough for REAL TIME PCR analysis for expression analysis

In our experience, digital droplet PCR is better than either -- by a wide margin.

REAL TIME PCR is specific for gene expression as it is more sensitive than bDNA.

Definitely real-time PCR, because it is more specific and accurate. I have expirience with both methods and now I preffer real-time PCR.

Have you ever used QuantiGene 2.0 Assay kit?

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Good work!

I think real time pcr is more specific