Hello folks,
I am trying to cut a specific RNA using RNAse H.
To do that I'm annealing an oligo which targets my RNA (annealing buffer TRIS 10mM, NaCl 50mM, 1mM EDTA) heating the mix (with 1.9ug total RNA and 0.1ug oligo plus 50ul annealing buffer) at 95ºC for 3 minutes and then cooling it at room temperature.
Then I added 10ul of RNAse H buffer 10x, 39ul of water (40ul for negative control) and 1ul of RNAse H (as indicated in the protocol), then I incubated the mix at 37ºC for 30 min (the protocol indicates 20 min but reading some papers I saw a lot of people incubating it for 30min-1hour).
To stop the reaction I added 1ul of 0.5M EDTA as indicated by the protocol, and then I added 500ul of trizol for purification.
I tried purification by both Direct-zol kit and using chloroform, isopropanol... But the yield I'm obtaining with both methods is ridiculously low (I got 1ug in the best scenario) and the 260/230 values in the nanodrop are all below 1, to summarize the curves are dirty and that doesn't happen when I purify other samples.
Then I did the RT and a PCR loading 15ng of template, I amplified correctly my targeted RNA and GAPDH in the INPUT (original RNA) but in the negative control my RNA appears at cicle 22-24 but I cannot see GAPDH until the cycle 28-30 (and with a low signal) and in the RNAse H treated RNA I obtain the opposite result, high signal for GAPDH (still lower than in the INPUT) and a low signal for my RNA.
I also checked the integrity of my RNA (unfortunately I only could use 200ng of RNA for the treated samples due to the low yield/quality) and even if the signal was low it wasn't degraded.
I hope I was clear and I wonder if someone else have had the same problem and how (s)he solved it or if you have any tips. I will keep doing another tries anyways.
Thank you!
- I am wondering why you are attempting to Digest a specific (Target) sequence?
- You just seem to be making matters more complicated than they need to be:
- In essence, in standard RT PCR or RT followed by Sybr green qPCR or qPCR from cDNA using Taq Man specific probes you
Laurence Stuart Dawkins-Hall Thank you for your answer. I'm digesting that specific target because it is a circRNA and I'm trying to open it in a specific place.
The RT-PCR is just to control that the digestion happened. Actually, I only need the RNA for direct RNA sequencing (and for that, I need a free 3' that is why I'm opening it).
I should have explained it better sorry!
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