In most cases, yes. But I suggest to use two or more control genes in RT-PCR, since some so-called housekeeping genes could have varied expression in some tissue or cell lines.

Thank you sir! Im working on houskeeping genes of archaebacteria. Is it ok to use just one if this is the case?

I used 16S RNA as a housekeeping gene with Yersinia enterocolitica and It worked very well. The two articles in which this housekeeping gene was used were:

- Inhibition of Quorum Sensing (QS) in Yersinia enterocolitica by an Orange Extract Rich in Glycosylated Flavanones.

- Urolithins, ellagitannin metabolites produced by colon microbiota, inhibit Quorum Sensing in Yersinia enterocolitica: Phenotypic response and associated molecular changes

If you need to know something do not hesitate to ask me.

Hi Mark,

As Zhi commented, 16s should be okay. However i would check if its expression changes along with your treatments, or time, or whatever variable is in your data set. If its expression do not change with your treatment, the you can be sure that you can use it as a good housekeeping gene. alternatively you might wanna try 18s, or some others from mitochondrial origin may be?.

I hope this helps, and goo luck!

thank you so much sir Yuanul, Zhi xi and sir Urbina! it really means a lot to me!

Hi,

16sRNA is good one. Like it is mentioned, the consistency of Ct values in various experiments, of your endogenous control should tell you how good your control is. Least variability with experimental treatments should keep the control unchanged in terms of fold differences. And if you ever want to use a protein coding gene, do take care whether it has a poly A. In lower organisms there might be small and stable poly-A RNA sometimes.

If your gene of interest has a poly A, be sure to use poly A specific primer/probe. Especially if you are looking at Taqman.

Kalpita

Thank you mam Karan!. im still new in the world of molecular analysis. im planning to quantify the protein product instead of of the gene. im pretty worried though because antibody synthesis takes months. any ideas?

Mark, Ab generation does take time. I think no shortcut. Protein product through western? Generating monoclonal or polyclonal?

Maam karan, not sure though on which type is appropriate for my study. I am quantifying the amount of a enzyme produced by a microorganism given this environmental factor. Would it have similar results if i quantify the mrna of instead of protein? Thank you maam.

Intensified gene's expression should lead to more pronounced protein presence; that's why it is intensified first place, right? ;)

Regarding your question of control, you DO need to use at least one more internal control, even better to run 3 independent normalization controls (tubulin, cox, you name it).

Uhuh ok..thank you sir..how many trials do you usually use in the quantification process? This is my first time to undertake such experiments so my knowledge and experience though is relatively young. Any help would be appreciated! Cheers.

Well sometimes higher gene expression doesn't mean more protein but more regulation at the level of RNA or regulating a proteins expression.

Actually monoclonal might be even more difficult to generate at the first go for its specificity and once at least a polyclonal Ab is generated and tested for specificity of your protein through western or whatever experiment you might plan to, then you would be able to say what is best.

Till you have an antibody, u cant predict. Yes the Antibody generated against the whole protein is far more specific than one generated using a peptide. But even generating through any peptide should be based on its uniqueness so that it doesn't have cross species reactivity.

Assuming MAC wants to quantify expression levels of a gene coding an enzyme, the relation's somewhat clear, right?

It's usually at least 3 technical reps (i.e. >=3 reps of one template per one primer pair), and at least 2 true biological replicas (independent experiments; in this case probably cultures taken one after another. You CAN mix biological reps on one plate though).

Your answers have been very helpful! thank you everyone!