I have ddPCR (digital droplet PCR) data from two different samples (control and unknown) and ddPCR data indicate that unknown sample have higher copy number/uL. I would like to know copy number/cell from these data. Is it possible to get this type of analysis using these data? Please let me know how if someone has expertization for this.
Thank you very much in advance
You can figure out the exact copy number if you have a standard curve.
But the point is you want to know copy number per cell, in best of my knowledge it's impossible.
We don't have two cells having same number of RNA at same time.
If anyone has a solution, I will glad to know.
Regards.
Jack Greenhouse thank you for your input. Could you please elaborate this protocol in depth so that I can performed this experiment precisely.
Hi,
There are several options to compare samples
- option 1. To compare several samples, you have to normalize your results towards the expression of reference genes (yes, plural). In a first experiment, you would have to assess the stability of several reference genes. Subsequently, you can pick some stable ones (yes, more then one) and use them for normalization. This allows you to assess for variation in Reverse Transcription. However, it does not tell you how much cells you've got.
- option 2. Selfie ddPCR ( https://www.nature.com/articles/s41598-017-08270-w ). Hereby you normalize towards gDNA copies of the same gene. So, it would allow you to 'determine the number of transcripts per gene'. 2 considerations should be made:
- The copy number of your gene (probably unknown). So, you would have to assess some reference genes on the gDNA level as well if you want to calculate it back to cells (also described in the paper).
- The fact that your RT is very likely not 100% efficient (probably rather inefficient). Using a different RT might give you different results. Furthermore, with this assay, you are not accounting for variation in RT-activity between samples.
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