Hi everyone,
I want to hybridize a 30nt-long poly dT on mRNA (which have polyA sequence). I extracted the total RNA and let it hybridize with a 5‘ FITC labelled polyT30 (heat to 95℃ for 5 minutes and cool to 25℃ in a rate of 0.1℃/s, stay for 2h). The results was shown as attached. The right two lanes are on the gel without sybr green I staining, so only fluorophore could be observed. The left two lanes are stained with sybr green I. As it shown, there are total RNA in the lane. However, I cannot observed any fluoresce labelled RNA. Why is that? Is it because of my electrophoresis condition? Mine is 1% agarose, 100V, 30min in TAE buffer.
Thank you,
Guangqi
Dear Guangqi,
your labelled T30 oligo likely hybridizes well to the polyA tails but:
1) the mRNAs in the total RNA pool are heterogenous in size which means that you won't see a discreet band
2) you likely added too much T30 oligo so that you can stil see the majority of it in the non-hybridized form
You can prepare samples with T30 oligo and minus and plus total RNA. If you quantify the T30 oligo bands in the lanes minus and plus total RNA you will see that in the plus RNA lane the band will likely be less intense. By comparing the intensities you can roughly estimate what fraction of your oligo got bound to the polyAs.
For basic biochemical studies you usually perform some titration experiments in which you mix two compounds of interest, one at a constant concentration and the other at varying concentrations. Why do not you try titration of your T30 untill it disapears as a non-hybridized band?
For RNA I would reccommed 70-80C instead of 95-100C and do not forget to eliminate divalent cations that lead to RNA cleavage at high temperatures. You can use a formulation with 20mM Tris 8.0 or 8.8 (the pH will drop in high temps by ca 0.027 per C) , 1mM EDTA (mg2+ chelation) and 50mM NaCl.
Best,
Zbig
Hi, Zbig!
Your suggestion is really informative! I tried taking image without the polyT band, just want to see if the mRNA has a fluorescent signal (even a little bit), but nothing observed as well. That's something that quite puzzle me actually. I used a GE Typhoon FLA9500 which I believe is quite sensitive.
Titration is a good idea. I will try and let you know the result, thank you very much.
The buffer currently I used is 1x PBS (pH 7.4~7.6, 0.3M NaCl, without Ca2+&Mg2+). I didn't take change in pH into account before. I will try your suggestion, thanks again.
Best regard,
Guangqi
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