regrading realtime-qPCR

[Raja Ishaq Nabi Khan]

Hi everyone,

I recently ran a splicing analysis using rMATS and identified some differentially spliced genes. I'm now trying to validate a few of the key ones through real-time qPCR, but I’m running into some challenges with confirming the splicing events.

If anyone has experience with this kind of validation and wouldn’t mind sharing some guidance or thoughts, I’d be really grateful. I’m still learning, and any help would mean a lot.

Thank you so much in advance!

[Theo]

Since you are not providing enough details I would only recommend to use the knowledge you got from rMATS, to design proper primers to cover the alternative splicing events.

In order to assess the magnitude (fold change/ΔΔCt) you will need primers that are unique for the splicing events, specific, and produce more or less the same amplicon size. treat your samples with DNAse. decide if polyT primers for reverse transcription are better than random primers.

I hope this helps as a starting point.

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[Thomas Danhorn]

To get a primer specific for a particular splice junction, make sure it is
right on top of it, so that the primer won't bind unless you have that
junction. There should be plenty of papers where this type of validation
has been done, so you can look at what they did. If you want to be 100%
sure that your PCR product is what you think it is, you can do Sanger
sequencing on it.

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