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Need help with my real time RT-PCR Plate Set up - (Jul/02/2013 )

I need help with my plate (96 well) set up for my real time RT-PCR

Goal of the experiment:
I wish to determine 4 different mRNA expression levels of 4 different viruses, during 4 different time points.
For simplicity I will call the viruses: V1, V2, V3, and V4
The mRNAs/Primers: A, B, C, and D
And the Time Points are: 1hr, 3hr, 6hr and 9hr
Controls: Mock infected, No Template Control (NTC), and No reverse transcriptase (NoRT)

I need my samples to be in triplicates; however, I am unsure what else I need to have in triplicates. I will normalize my values against GAPDH, therefore, GAPDH should also be in triplicates,,,right? Should I also run my NoRT and NTC in triplicates as well?
Also I have to separate my 96 well plates by primer.

Thanks for any help!!


First question is, what are you going to compare together?
All of the timepoints and viruses together or just all viruses within same timepoint or what.
The main rule is all samples compared together must run within same plate. Different primers may be on different plates.

You should run everything in triplicates, target genes, GAPDH, all controls.

But I need to understand better the setting, I have problems to calculate it. Does each virus RNA correspond with one of the primers? Like A for V1, B for V2.... or you have 4 different primer pair for each virus (that would be strange since you only mention 4 primers and you say viruses are different, it's unlikely they would have same mRNA sequence). I need to know how many samples and how many genes run on them, overall.

So how many cDNAs you have, five (four timepoints plus mock, or does mock have timepoints too?), and the NoRT is for every cDNA made or just once? (in that case it's not ideal NoRT control, just a quick check).


Thanks for your response, sorry I am getting back to this so late. I'm also sorry about not explaining myself clearly.
The viruses are all the same (Sendai virus), just different variants.

<*> what are you going to compare together? - I'm comparing the infected samples ( each of the 4 virus variants) during the four time points.

I have constructed my plate set up by primer. For example, each of the viruses have the M gene, (plate #1), I was only able to fit everything on one plate when I did duplicates of each virus at each time point, alongside each virus sample I placed a noRT (reverse transcriptase) control. I also ran a mock and a NTC with RT and no RT.

check out this pictures for a visual:

I ran the experiment (for only one primer yesterday) and I got pretty good results!


So from the pictures.. you have noRT for each cDNA sample (not for mock? is the "mock infected" also a real cell sample? in that case it should have noRT control, unlike the NTC which is supposed to be just water instead of any DNA so it has no logic to have noRT for NTC).
Duplicates may be fine if there are not big differences, but if there are, you have the disadvantage that you don't know which of the values is off. In triplicates if one value differ, you can tell from the other two, that are close by, which value is probably off, and you can even discard it if needed.

And as for the comparison, still not clear, sorry, maybe I didn't ask well enough.
Question is if you are using relative quantification equation for this and if so, what is your calibrator then. Calibrator is a sample to which all others are compared. For example a timepoint 0, if you have several timepoints or "untreated" sample if you have several treatments. It's not clear in this virus setting, that's why I'm asking.

Whether you chose one virus as a calibrator in each timepoint (like, values of virus RGVO will be 100% for each timepoint separately and the others viruses would be relative to that - that is if you want to compare viruses with each other at given timepoint) or you will use a timepoint as a calibrator for each single virus (like values from 1hr timpoint will be 100% and values for other timepoints for each virus separately will be compared to that - that is is you want to compare changes in expression in time for each virus separately). In these cases there are more calibrators, and only samples that use a given calibrator has to be at the same plate with it. So you could divide your plate according to that.

Or of course you may chose just a single calibrator for the whole set, one of the viruses at set timepoint, but those data are not comparable together very well I would think.


The Mock is uninfected, originally I thought it needed a no RT; but my PI just thought it might be over kill and decided that I should save the reagents. After thinking about it again, I agree, the no RT for the NTC is not logical, I probably will not include it on my next plate.
I am using the relative quantification equation; however, I am still not too sure what my calibrator will be. In the past they used one of the wild type viruses, which I believe makes sense, as the goal of this experiment is to elucidate why one of the virus variants is not producing infectious virions in the same manner as the wild type.
I would like to run my templates in triplicates but I'm afraid I don't have the room, would you suggest using multiple plates? I'm sure I can do triplicates if I run two plates per primer, but I'm wondering if the error between plates will mess up my analysis.
Thanks for the help!


The complete analysis scheme has to be decided before you set up any reactions! That is a common mistake I'm seeing.

First you need to sit down and think what is going to be a calibrator, or if you have more possibilities consider them all and then start the plate design.
Because that depends on the calibrator used.

For example if you have a timepoint as a calibrator, you only need to include other timepoints at the same plate (in triplicates) if all your samples won't fit at once. That way the inter-plate error is put of question since all you are comparing together is on the same plate.
If you still have more samples than fits, you simply include the calibrator sample again on the new plate and since both are the same calibrators, you will calculate with the first-plate calibrator values for samples on the first plate and with second-calibrator values for the samples on the second plate.(include NTC for every separate master mix you make, if you pipett both plates from a single mix, you only need one)

This is the reason why you need to be sure about your calibrator(s) before reaction set up, becase you need it for decision on the plates layout.


I see, well if my calibrator is wt-F(which I'm almost positive it is) my plate set up should be fine,, right?


So your calibrator is wt-F in all timepoints? Like you are interested in differences between virus variants within one timepoint? So wt-F value in each timepoint will be 1 (or 100%) and other viruses relative to that?

Or you in addition want to see changes of each of the virus in time too. Because these could be two different analysis, done from the same dataset, depending on if you want to analyse several things. You may want both, in that case you need all samples on one plate or with all respective calibrators or of course run it twice each time with different layout.
But maybe that is already known and not important, this is really a thing to be decided by the scientist involved because no one else can, without seeing the whole picture of the project, say what exactly are you trying to prove or find.

In your case, you can do any analysis from your layout, because you have all samples for single gene on one plate, but on the expense of using only duplicates.


Yes, (RE using wt-F = 1) from what I have read from a previous masters student's work on this project. They used wt-F as the calibrator in all the time points, although the work was not done in duplicates or triplicates, but in singles. The values generated between viruses and their respective mRNAs were indeed relative to wt-F. I still need to inquire more about why this virus (wt-F) was chosen as the calibrator, as I am a little confused about that now lol thanks!

as for the question about "wanting to see change of each of the viruses in time" I'm not too sure I understand what you mean by this? Thinking about gaining another analysis from the same data set is pretty interesting though!

In a Nut Shell: The project aims to measure the expression of retaliative amounts of different viral mRNA, between Sendai virus variants (the variants have specific mutations which will help determine molecular mechanisms involved in viral characteristics), at the early stages of an infection: 1, 3, 6, and 9hrs (done in LLC-MK2 cells). One of the variants produces an atypical lethal systemic infection, and one of the variants is becoming attenuated. Discovering the molecular mechanisms involved in attenuation, or virulence for that matter, can help in either prophylaxis or post exposure treatments of viral respiratory infections.

ps: thanks again for the help