Basic question on collecting liver for RT-PCR - Never did this before, won't get to do this again with these anima (Jan/21/2008 )
Hello. First of all, I wasn't sure whether to put this under RT-PCR or general lab techniques, because my question is actually about obtaining tissue for subsequent isolation with Trizol and RT-PCR. If I'm posting in the wrong sub-forum, I apologize.
Anyway, I have these kinda-precious mice that I only get one shot at, and I've never prepared tissues for RT-PCR before. The tissue I'm collecting is liver, and the purpose of the RT-PCR is to follow up on genes identified in a microarray study.
- First of all, is it better to snap-freeze livers or to put them into RNAlater? Or something else altogether?
- Secondly, how important is it to fast the animals before collecting livers? If it's standard practice to fast them, how long do people do so? Can anybody recommend a primary reference on fasting in this context? I should say that the genes I'm looking at have no direct relationship to insulin signaling, nutritional state, etc. Rather they are in the AP1 and NFkB pathways.
- Thirdly, is there anything you wish you knew when *you* did your first RNA isolation for RT-PCR that you found out the hard way? :-D
Thank you kindly.
Anyway, I have these kinda-precious mice that I only get one shot at, and I've never prepared tissues for RT-PCR before. The tissue I'm collecting is liver, and the purpose of the RT-PCR is to follow up on genes identified in a microarray study.
- First of all, is it better to snap-freeze livers or to put them into RNAlater? Or something else altogether?
- Secondly, how important is it to fast the animals before collecting livers? If it's standard practice to fast them, how long do people do so? Can anybody recommend a primary reference on fasting in this context? I should say that the genes I'm looking at have no direct relationship to insulin signaling, nutritional state, etc. Rather they are in the AP1 and NFkB pathways.
- Thirdly, is there anything you wish you knew when *you* did your first RNA isolation for RT-PCR that you found out the hard way? :-D
In the past I have snap-frozen the liver as soon as possible after sacrificing the mouse. You only need a small bit of liver to get enough RNA so you'll have bucket loads if things don't work out the first time! After you homogenise the tissue in trizol , make sure you spin the get rid of any crud and then use the supernatant for the rest of the procedure. I was got a result with RT-PCR although the PCRs were never as "clean" as other organs. Perhaps someone elsecan advise?
I have found that RNAlater works much better than liquid nitrogen frozen tissues. We harvest tissues from maternal and fetal organs so the necropsy time is a little longer which is why I believe RNAlater works much better. The liquid nitrogen frozen tissues often have degraded RNA compared to fresh samples stored in RNAlater. In addition, RNA later tissues are stable and pliable (easy to cut into smaller sections) at room temperature while liquid nitrogen frozen samples are not.
1. We also have good experience with RNAlater regarding RNA stability.
3. Be sure to get a high quality RNA and DNA-free. You can't get completely DNA-free RNA from Trizol, so we do DNase treatment before reverse transcription.
And if you're doing all these things for a first time I would personaly do a complete isolation on some other sample, to try everything first. You will need a control anyway.