Hi, I just looked another similar study "(A Novel Strategy for Selection and Validation of Reference Genes in Dynamic Multidimensional Experimental Design in Yeast(Cankorur-Cetinkaya A, Dereli E, Eraslan S, Karabekmez E, Dikicioglu D, et al. (2012) A Novel Strategy for Selection and Validation of Reference Genes in Dynamic Multidimensional Experimental Design in Yeast. PLoS ONE 7(6): e38351. doi:10.1371/journal.pone.0038351)" but the genes I mentioned above are nowhere in the list of stable reference genes reported in the paper mentioned in my original question. My target genes are quite weakly expressed and it is really important for me to get a handle on this reference gene issue asap. Please help....

I always used ACT1 for my cDNA qPCRS. It is highly and constitutively expressed under almost all conditions. Since most genes are expressed at much lower levels than ACT1, I used 10x less template for ACT1 qPCR reactions. Hope this helps..

Hi

I used ACT1 for S. cerevisiae cDNA and studied under oxidising conditions by qPCR analysis. This gene worked well for me. U can also try.

Best wishes

Hi Bilge, did you validate the expression of ACT for your samples? As I mentioned earlier, my target genes are very weakly expressed and even the slightest variations in the expression of the reference genes can cause major fluctuations in the expression profile of my target.

Thanks

I haven't done a thorough analysis but the absolute cts were pretty stable from one condition to another. It is also widely accepted in the scientific community as a reference gene in cerevisiae both for its stable mRNA and protein levels.

I use ACT1 too.

Dear Syed,

I understand your trouble if the results that you obtained with these to putative reference genes do not perfectly match. However, these are ‘putative’ ref genes that need to be certified in YOUR assay. And I am not surprised if one of them (but which one, ALG9 or TFC1 ?!) would not be OK. We must really keep in mind that there is no single universal gene that exhibits stable expression levels in any sample and/or organism of interest. Including the ACT1… The need for systematic validation of the stability of transcript levels from these reference genes in any new study is absolutely warranted. I therefore encourage you to try other genes from the list proposed in the BMC Mol Biol paper, including UBC6 that proved to be very encouraging in our last study*. And of course, do not do the mistake to use only one of them for normalization, but average at least three of them for final normalization of your GOIs. At least three, and may be more if you think/prove that there is some substantial variation.

About the ACT1, test it if you want. But compare its stability to the other putative ref genes with the appropriate program. And you will see, in YOUR assay, whether you can keep it -together with 2 or three more ref genes-, for normalization.

* We are waiting the publication of another paper about ref genes for normalization in fungi (BMC Genomics, under revision and coming soon… hopefully). Again, by using an exhaustive set of publicly available data (almost 100 very different conditions (growth , development, stress, etc), from about 20 different fungi ), we could prove that the ACT was not the best candidate … It can be OK in some specific conditions, but ‘statistically’ speaking, the risk to use this gene while it is not stable highly increases. To answer Bilge’s comment, if it is true, I think that it is nowadays regrettable that the scientific community continues accepting ACT1 as a reference gene in cerevisiae (or any other organism) both for its stable mRNA and protein levels.

Thanks Jean for the elaborate answer. I was really worried if my concern regarding the reference gene was really that critical or not. Since everyone I talked to about the problem seemed to have never encountered a similar issue with their study.

One more  precision. To assess the stability of the ref genes that you will evaluate in your study, and help you choosing the best ones to normalize your GOIs, follow this link :  RefFinder (http://www.leonxie.com/referencegene.php). THis is a web-based tool that integrates the currently available major computational programs (geNorm , Normfinder , BestKeeper and the comparative ΔCt method). Its use is very simple.  Good luck. JL

Hi,

I have recently done some qPCR after total RNA extraction, followed by first strand cDNA synthesis. The problem is, while one treatment is giving me closely lying values for relative gene expression, the data points for the other treatments are much widely dispersed. Following is the box plot showing all the data points for each treatment.

Two reference genes are used (Reference1 and Reference2) as the internal reference. These reference have already been validated and found to be expressed at a stable level across the samples.I am really concerned about the spread in treatment 3-5. Shouldn't the dots in these treatments be as tightly grouped together as they are in treatment 2 or in treatment 1 (if the two outlying data points are ignored)?

Hi Jean-Luc Parrou,

I was wondering why did you use a forward primer for the ACT1 that lies on both the intron and the exon, rather than one that is only located on the exon? Have you tested both options?

Thanks!