- Joined
- Jul 17, 2003
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Hey everyone,
Since all of you MSTPers are super well-versed in lab and lab techniques, I hope you don't mind me venturing in from pre-allo and asking you for some tips on RNA isolation. I'm sure it is something many of you have done a million times, but I'm doing it for my first time and I'm having some issues.
Specifically, I am talking about RNA extraction from tissue (mouse lung). We harvested the mouse lung and snap froze it in smaller pieces. I pulverized the lung while frozen and sonnicated/homogenized it with RNA Stat-60 and proceeded with the isolation. When I sent it up to the Core facility to check the 28S: 18S ratio with the agilent bioanalyzer, the RNA was all degraded.
OK, so I thought, maybe its cuz I'm doing it for the first time and my technique is contamination-prone (which I didn't think it was). I did it again with another person in lab who is more well versed with RNA isolation (usually from cells though), and the RNA was still degraded. I was super vigilant in making sure I wasn't contamination-prone. But still... degraded RNA. (Oh yeah, concentrations look ok, as does the 260/280 nm ratio).
what do you guys suggest? a Qiagen prep kit? use a glass homogenizer instead of the sonnication probe? Am I overlooking some key part? Am I just not getting it?
I really want to get this right so I can move on to the cDNA and real time PCR part of the project. I feel stuck on this step, and I don't know what my next move should be in attempting this isolation.
Thanks for reading this post, sorry it was so long. I feel like this is something that is fairly easy for people, but I'm the loser who can't get it right! 😳
Lux
Since all of you MSTPers are super well-versed in lab and lab techniques, I hope you don't mind me venturing in from pre-allo and asking you for some tips on RNA isolation. I'm sure it is something many of you have done a million times, but I'm doing it for my first time and I'm having some issues.
Specifically, I am talking about RNA extraction from tissue (mouse lung). We harvested the mouse lung and snap froze it in smaller pieces. I pulverized the lung while frozen and sonnicated/homogenized it with RNA Stat-60 and proceeded with the isolation. When I sent it up to the Core facility to check the 28S: 18S ratio with the agilent bioanalyzer, the RNA was all degraded.
OK, so I thought, maybe its cuz I'm doing it for the first time and my technique is contamination-prone (which I didn't think it was). I did it again with another person in lab who is more well versed with RNA isolation (usually from cells though), and the RNA was still degraded. I was super vigilant in making sure I wasn't contamination-prone. But still... degraded RNA. (Oh yeah, concentrations look ok, as does the 260/280 nm ratio).
what do you guys suggest? a Qiagen prep kit? use a glass homogenizer instead of the sonnication probe? Am I overlooking some key part? Am I just not getting it?
I really want to get this right so I can move on to the cDNA and real time PCR part of the project. I feel stuck on this step, and I don't know what my next move should be in attempting this isolation.
Thanks for reading this post, sorry it was so long. I feel like this is something that is fairly easy for people, but I'm the loser who can't get it right! 😳
Lux