RNA extraction using Trizol - (Dec/17/2003 )
I am trying to amplify a 900 bp fragment from cDNA using PFU Turbo. I created the cDNA through RT-PCR of RNA that I extracted with Trizol. I am able to amplify a 400 bp fragment from the B-actin gene; however, I am unsuccesful in amplifying a larger 900 bp fragment. Does he problem lie in the fact that I have fragmented RNA and as a result cannot amplify this larger fragment? Can Trizol be used when you need to amplify a large fragment?
I don't think RNA fragmentation during isolation is the problem. Are you sure the sample you used for RNA extraction expresses the gene? How long did your RT run? If you want to get longer cDNA, run your RT a little bit longer, 45 min to 1 hr is enough for 900 bp.
oh man have i had some problems with trizol! i've also tried similar experiments and for some reason get similar results. for my stuff, it appears that the smaller your RNA yeilds, the poorer the quality of RNA when you use trizol. another problem i've had is when i quantitate my RNA on the spectophotometer after trizol extraction i end up with a value, and when i work with that concentration i find that it's totally inaccurate. it sounds strange, but i always have less RNA than i calculate after the quantitation. this never happens when i use alternative RNA isolation protocols. so if your gene is expressed at low levels normally, maybe your quantitation is also out and so there's not enough transcript to PCR from - but because b-actin is expressed highly you can still pick it up? like i said - sounds strange, but it's happened to me. consequently i never use trizol anymore.
hope this helps...