I have two biotins--one of which is binding my RNA with cleavable bonds, and the other with irreversible bonds. Does biotin have an effect if it keeps its bonds with RNA? If it does, why is this the case?


Having looked into this question, I came across this paper, where they mixed biotin labelled EGFR-mutant-specific oligonucleotide and isolated the target using streptavidin beads followed by real-time TaqMan PCR allowing a 40 fold increase in detection sensitivity of the mutant allele, suggesting that biotin does not effect nucleotides in PCR based experiments.

The life technologies webpage claims that Streptavidin-Coupled Dynabeads M-270 can be used in a PCR reaction as stated below although certain streptavidin-coupled beads can have an effect on the PCR reaction. Hence I do not think biotin itself would be a problem for an amplification reaction.

beads can be used directly in PCR, as they do not inhibit enzymatic reaction. Slight PCR-inhibition has been seen when 75–100 µg of Dynabeads® M-280 Streptavidin was used in 50 µL PCR reaction, and a concentration over 100 µg per 50 µL reaction seems to inhibit PCR completely. Dynabeads® M-270 Streptavidin do not show any inhibitory effect on PCR at these concentrations.

In fact you can buy kits such as this where you can generate/incorporate biotin into your RNA of interest for any purpose. The use of biotin and its incorporation into nucleotides seems prevalent in the literature and there are even kits for this purpose hence you should be on the safe side with your biotin labelled RNA experiments. I think the biggest challenge in RNA work is to keep it from degradation, which as far as I know is achieved through maintenance at -80 oC.


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