DIY RNA Spike-in controls for qRTPCR
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5.2 years ago

I'm hoping to find a low-cost alternative to the ERCC spike-in controls for use in qRTPCR. Due to a high concentration of lipids in my tissues, RNA extraction between samples can be variable. I'm hoping to use something like the ERCC or TATTAA spike-in controls to normalize differences between sample prep. Ideally, I would use 3-5 synthetic RNA oligos to spike in.

Both the commercial spike-ins are pretty pricey, and more extensive than what I need. Has anyone had success with their own do-it-yourself (DIY) spike-ins? Did you buy synthetic RNA oligos from a company and then poly(A)-tail and 5'-cap them yourself? Are there companies that will do the tailing and capping in addition to oligo synthesis?

Thanks for any advice!

qpcr rtpcr spike-in rna • 3.2k views
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I haven't done this, but I would suggest thinking about ordering DNA oligo's in which a T7 promoter is included, as such you can perform in vitro transcription yourself. This would probably be cheaper and your DNA is more stable...

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I think you could try luciferase control RNA from Promega. It is not completely DYI, but sequence is known (if you will be interested I'll try to find an article with that) so one could construct primers and order it separately, what makes it not very expensive.

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Interesting idea! An article link would be greatly appreciated!

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Here it is:

An Internal Reference Technique for Accurately Quantifying Specific mRNAs by Real-Time PCR with Application to the tceA Reductive Dehalogenase Gene. David R. Johnson, Patrick K. H. Lee, Victor F. Holmes, and Lisa Alvarez-Cohen

http://dx.doi.org/10.1128%2FAEM.71.7.3866-3871.2005

I've used it and it gives really good results.

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Great! Thanks so much!

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