Hey everyone,
I am working on liver tissue samples. I have FACS sorted 3 populations from same tissue and I want to study the gene expression between these populations using qPCR.
One of the population has sufficient total RNA while other two have very less amount of total RNA (49 ng/ul- elution volume 25 ul). My question is, why it is important to take equal amount of total RNA for cDNA preparation? Suppose we take 800ng total RNA from one sample and 700ng total RNA from other two samples each, and dilute both to 5ng/ul for qPCR reaction, will it significantly affect the gene expression? Also, I generally use at least two genes to normalise my gene expression results by 2^(-delta ct) method. But please give your expert opinion and if possible a reference to support your answer.
Best regards and thanks,
Ankur
Hello Ankur,
In my experience, a difference of 100ng RNA in the reverse transcription reaction probably won't make that much of a difference for the qPCR in the end. Generally, using similar amounts of starting material will help to increase the comparability of your samples - this actually applies to so many methods (I'm thinking of loading agarose gels, Western Blot, ...).
And adjusting samples in the very first step is the simplest thing to do, right?
To minimise irregularities in your qPCR data that may have arisen simply through the method itself (e.g. less efficient amplification of low-expression targets...), but do not reflect actual transcriptional changes, it is desirable to load similar amounts of cDNA. This will give similar Ct values for the housekeeper you use for normalisation.
Using two housekeepers is always a good idea, there are also tissue-specific / cell type-specific recommendations on non-regulated targets that make for reliable housekeepers. I would assume that especially liver tissue as a key player in metabolism displays active regulation of numerous targets that are not regulated in "less active" tissues. So, selecting the appropriate housekeeper for your approach should be done with care!
Some (quickly gathered) examples from the literature available on liver tissue qPCR - there is a lot more out there!:
https://www.nature.com/articles/srep38513
Article Validation of control genes and a standardised protocol for ...
Article Housekeeping Gene Variability in the Liver of Alcoholic Patients
Article Selection of reference genes for quantitative RT-PCR (RT-qPC...
All the best,
Katharina
Hello Katharina,
Thanks for your time and effort to address my question. I agree with your point that keeping things as similar as possible at the beginning will help to reduce the anomalies, so maybe I should scale down the abundant sample to meet the level of scarce samples.
I am happy to know you address the issue of liver specific marker or housekeeping genes because I recently found about it and yes I have incorporated this hence I am using genes which is expressed at constant level throughout liver like Ywhaz and Ap3d1 and as you are also working on liver, I would love to know about the controls that you are using.
Thanks for the links I will go through them at the earliest possible.
Best regards,
Ankur
Hey Ankur,
I am happy that I was helpful!
At the moment, I am working exclusively with a hepatoma cell line; for this, I have found GAPDH to be good enough, although I found some stressors which can cause GAPDH regulation. In that case, betaActin will be my choice. Apart from that, I am a big fan of 18S rRNA! As far as I know, it works well for many different cell types.
Reverse transcriptase activity is unequal to two different quantities of RNA input, which ultimately reflects altered expression of our gene of interests.
Thanks Katharina and Chandra!
So I asked this question to my Prof. He explained me that if I take two different concentrations, the amount of transcripts between two samples might differ and specially for lower transcripts this might induce a bias. Hence, it is advisable to use equal amount of starting material.
I really appreciate your feedbacks!
Thanks again!
Actually you are checking the expression level of genes in two or more samples. If you take different concentrations so you are risking the transcripts number per sample. Your teacher is absolutely right.
I agree with the ansewers. If you start with different concentration it affect the result as the most concentrated sampes will have more copies of your gene, that you might wrongly conclude as high expression level of that gene while it is not the case.
Good Luck!
Your prof. is right. I would be careful to use the same amount of starting material even if in reality they are not exactly equal amounts (very insignificant though). You may choose your gene of choice to normalize your run based on your experience or what has worked for you. however you should be able to use one that does not change across samples, treatments, etc. Reviewers will give you a hard time on that. RPL8 can be one of your additions. I always carry out pre-runs whenever I have to work with a different organism.
It’s not mandatory but it’s suggested. In my opinion besides GAPDH, low expressor TBP is also a good endogenous control as you get good feel of how those low expressors might be amplifying. As far as amount of RNA is concerned imagine you have 1ng of one sample and 10ng of other the GAPDH and your gene of interest will show lower Ct value in high conc sample but the delta ct will remain constant assuming 100% PCR efficiency. So, in conclusion results will remain same.
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