Very late CP for control in rt-qPCR from RNAlater samples - (Jan/03/2012 )
I've been having problems with my rt qPCR lately. Let me explain the details of how I did my experiments. Im working with adult pancreas with contains lots of nucleases so we used (for the first time) RNAlater.
Upon dissection I placed the pieces of pancreas in RNAlater and kept O/N at 4°C, then I took the pieces out removed the RNAlater and disrupt them in triReagent (trizol) and did the extraction as usual with phenol/chloroform and O/N isopropanol precipitation.
The RNA obtained is concentrated at around 1.5ug/ul and the ratios are good (260/230=1.8 and 260/280=2). I took 4ug for the DNAseI treatment and the RT using Transcriptor Reverse Transcriptase from Roche.
I then used 1.5ul of the RT with 8.5ul of the mix (containing probe, primers and LightCycle480 mix) as we usually do on embryonic tissue.
However, when I look at the result of my qPCR, the internal control gene that we use (Rplp0) has very late CP of about 30 (LightCycler480 machine) normally it comes out between 21-25 cycles.
I thought the problem could be the RT and I repeated it but the results are similar with a new batch of Transcriptor Reverse Transcriptase.
Does anyone has experiement with RT using RNAlater treated samples? What would be your suggestions to make this work better?
Thanks for your help and happy new year.
Here you are dealing with two issues, one is the inherent inhibition from RNAlater reagents, you can check it out by titrating the RNA amount you put in for RT, if there are impurities in your RNA, you will het later Ct from more input RNA. If that's the case, using less RNA or even diluted RNA will be beneficial to you. Next time, you may want to avoid using RNAlater, instead, try using LyseNow kit from Metammune or RNASound additive for your homebrew tissue lysis buffer to stabilize your RNA; second issue is the high content of RNase in pancreas, RNA might be degraded already, for that, random hexamer as RT primer will rescue the Ct issue. Good luck!
Do you have any evidence of RNAlater being notably inhibiting RT? Because we use RNAlater-stabilised tissues and don't see anything like that.
I would personally doubt that it can have such effect, if you don't carryover significant amount of solution to Trizol, when you spin and wash the sample through isolation several times.
we used RNAlater as suggested by other teams working with adult pancreas.
I dont think I carried over RNAlater and if I did a little it would be gone in the numerous washes.
For the RT we already use random hexamer.
I redid the experiment with different RT enzymes, and asked someone to do it for me to avoid a problem of contamination by RNAse in my buffers or else.
The problem is still the same... I guess something is inhibiting the RT.