I do not have Ct values for housekeeper genes, as those may not be present in the sample and including them would be misleading. I need to be able to compare Ct values without housekeeping normalization.
Daniel
I would proceed with caution with what you are proposing and would not recommend it. For starters is you wish to publish such data in any reputable journal it is likely it would be turned down flat. Just a word of caution. Moreover as mentioned by Jack based on compliance rules (for publication) even 1 x housekeeping gene might not suffice for publication out put
Another caveat to keep in mind is that although Ct values for different genes from the same sample are theoretically plausible as comparative bench marks comparing expression even of the same gene from different samples or biological replicates is fraught given that different samples even derived from the same RNA will have different cDNA copy numbers, by virtue of purity differences, and without doubt this will apply to cDNA made from different RNA samples where the efficiency of RT will definitely differ. This is the reason for normalising with a housekeeper to correct for such efficiency differences
Thus, although in principle it might be possible to compare the expression of 2 different genes from the same cDNA sample based on Ct values alone I would strongly advise against not using housekeepers if at some point you wish to declare such data in a paper: It is likely that 1 or more of the above objections would be made and in any case reviewers could just turn around and say 'why not' ? Moreover, there are multiple high copy number stable genes for every situation I am aware of so I cannot comment on your assertion that no housekeeper is available for your sample
Reference gene normalization correct for potential differences of overall DNA or RNA concentration or integrity among your samples, so it is highly recommended to use at least one, and possibly more, carefully chosen reference genes. Other method for normalization can be used but are not so accurate (see Erickson et al. below)
Directly comparing your Ct values is of course mathematically possible but might be biologically flawed, if RNA concentration or sample quality is biased according to one of your biological variables of interest (experimental groups or other important covariates).
Could you please explain why references genes might not be present in your sample and be misleading in your case?
Article The MIQE Guidelines: minimum Information for Publication of ...
Article Assessment of normalization strategies for quantitative RT-P...
Loading the exact same amount of nucleic acid material per each qPCReaction is one way to tease out some meaning from Cq values in the absence of a reference gene. Although fraction of ribosomal RNA vs mRNA can theoretically be a caveat in this approach (see Vandesompele et al., 2002 regarding a cancer cell type), this approach has worked quite nicely in many other situations. Vandesompele et al., admit that the ideal reference gene does not exist, and the MIQE Guidelines may allude to 2 or 3 reference genes being necessary in most situations, but another reality is, that with enough careful preparation and precision technique, real results can indeed be gleaned from qPCR results in the absence of a reference gene. But, proceed at your own risk, and be extremely careful with spec and/or NanoDrop readings of your RNA samples.
Daniel
I would proceed with caution with what you are proposing and would not recommend it. For starters is you wish to publish such data in any reputable journal it is likely it would be turned down flat. Just a word of caution. Moreover as mentioned by Jack based on compliance rules (for publication) even 1 x housekeeping gene might not suffice for publication out put
Another caveat to keep in mind is that although Ct values for different genes from the same sample are theoretically plausible as comparative bench marks comparing expression even of the same gene from different samples or biological replicates is fraught given that different samples even derived from the same RNA will have different cDNA copy numbers, by virtue of purity differences, and without doubt this will apply to cDNA made from different RNA samples where the efficiency of RT will definitely differ. This is the reason for normalising with a housekeeper to correct for such efficiency differences
Thus, although in principle it might be possible to compare the expression of 2 different genes from the same cDNA sample based on Ct values alone I would strongly advise against not using housekeepers if at some point you wish to declare such data in a paper: It is likely that 1 or more of the above objections would be made and in any case reviewers could just turn around and say 'why not' ? Moreover, there are multiple high copy number stable genes for every situation I am aware of so I cannot comment on your assertion that no housekeeper is available for your sample
Dr. Hall has nicely summed up what more can lie under the tip of the iceberg here.
- Badges
- Science method