Hi Jose,

Apart from the other packagese like Genorm and Normfinder, Bestkeeper is not specifically made to construct a hierarchy of reference genes. Consequently the interpretation is indeed not as straightforward. You mainly have to assess the correlattion factors (the closer to 1 the better) and the standard deviation (the bigger the worse).

I would be cautious working with the software Sabine suggested. First of all, it doesn't take efficiencies into account, as the input is raw Cq values, whereas the original software packages all require efficiencies, or processed relative expression values. This might slightly change the outcome of the best reference gene. Second the order of most stable to least stable reference gene is made by comparing the different outcomes of the four methods arithmetically (Genorm, Normfinder, Bestkeeper and delta CT). These methods are all based on different algorithms and assumptions, so a straightforward comparison of their output is not possible. Common sense and a good knowledge of the underlying assumptions will help you best.

In addition, the ranking of the Bestkeeper results in the on-line tool is only based on the standard deviations, not on the correlations. These thus give a biased outcome as well.

Hey!

I recommend using this:

http://www.leonxie.com/referencegene.php?type=reference

Best,

Sabina

Thanks a lot Sabina !!!

Hi Jose,

Apart from the other packagese like Genorm and Normfinder, Bestkeeper is not specifically made to construct a hierarchy of reference genes. Consequently the interpretation is indeed not as straightforward. You mainly have to assess the correlattion factors (the closer to 1 the better) and the standard deviation (the bigger the worse).

I would be cautious working with the software Sabine suggested. First of all, it doesn't take efficiencies into account, as the input is raw Cq values, whereas the original software packages all require efficiencies, or processed relative expression values. This might slightly change the outcome of the best reference gene. Second the order of most stable to least stable reference gene is made by comparing the different outcomes of the four methods arithmetically (Genorm, Normfinder, Bestkeeper and delta CT). These methods are all based on different algorithms and assumptions, so a straightforward comparison of their output is not possible. Common sense and a good knowledge of the underlying assumptions will help you best.

In addition, the ranking of the Bestkeeper results in the on-line tool is only based on the standard deviations, not on the correlations. These thus give a biased outcome as well.

Ward,

Thanks a lot for your answer and explanation. I agreed with you. Initially I was thinking to use just Normfinder and GeNorm, however, I have seen that in some papers Bestkeeper is also used. After your comments, I am thinking to use bestkeepr just like an extra approach and focus my analysis in GeNorm and Normfinder outputs.

Thanks

Sabina, Jose, Ward,

Thanks for the explanations and link.

Just a questions to Ward: RefFinder takes raw Ct values and does not consider efficiencies. What is wrong with adjusting the efficiency manually (say using Genex or even Excel) to obtain adjusted CT values for input into RefFinder? Would that compromise the analysis?

Thanks/, Aman

Using adjusted Cq values may indeed resolve that issue, but it wouldn't solve the issues with the final ranking.

For your information, in my experience, I've found genorm, bestkeeper and normfinder to generate almost the same answer.

Thanks a lot, Sabina :)

hello there 

how can i enter my data on above link(the file format)

Do you mean this link http://www.leonxie.com/referencegene.php?type=reference ? If so, click "Try example" and set data in Excel. 

Unfortuntely the domain for  already mentioned site has been expired!!

RefFinder online does not work 

A new web of RefFinder

http://fulxie.0fees.us/?type=reference

Thanks Gunnaporn !

Thanks for the question and answers.

It helped me too...

The input in Refinder should be only the patient sample or  should I add the control sample?

Hi Friends

Based on this sentence in Pfaffl paper :

These x-fold regulation results are corrected via the factor specific real-time PCR efficiency, calculated according Equation (3).

Is  "std dev [± x-fold]"  related to primer efficiency that got from standard curve?

Thanks.

How to get access to RefFinder?

Hi All,

I see that the old link to Reffinder is erroneous. Do you know the new link ?

Thanks,

Hi, 

The link has been expired but I found sth interesting which refers to  Reffinder. According to the attached PDF this software gives biased output as PCR efficiencies are not taken into account.

What do you think?

New link for RefFinder is here...

http://150.216.56.64/referencegene.php?type=reference

thanks

Seyedeh Nahid Fotuhi Indeed, PCR amplification efficiency should be taken into account by Ruijter 2009. Thus Accugen software "RealCount" had incorporated amplification efficiency into calculation.

I've posted a new link to RefFinder in another post, here it is: https://www.heartcure.com.au/for-researchers/

Ankush Saddhe Eric Neau Samiullah Khan

Dear Terry

Is RefFinder free?

Thanks

Jose

Jose Manuel Latorre Estivalis yes it is and it combines 4 different methods together

Jose Manuel Latorre Estivalis

I have used RefFinder using the following link www.accugen.com.au/software suggested by Terry Lee . I found it as reliable and user friendly.

Thanks

Terry Lee Can I use 2-3 house keeping gene as reference gene to analyse my real time PCR data? If yes please explain how to do it

Nuzaiba Muhammed are you asking about how many reference genes are required for publication? MIQE guideline mentioned 1 reference gene is not acceptable. Most paper publish at least 2 reference genes. Here's a video for your question: https://www.youtube.com/watch?v=wJdE6wyhu58

thank you very much Terry, you are the best

just an update, I've changed the hosting, so the link is different now