I am new to the forum and in need of your expertise please.
I am trying to amplify a pcr product (using pfu pol) from cDNA that I have RT from RNA isolated from human breast cancer tissue on slides. As the amount of tissue I have available is limited I used a picopure RNA isolation kit. I am having major problems getting a product from the PCR and just a get a large smear on the gel. I have tried alter and optimising many things and Iíll try and talk you through what I have determined (sorry this may be quite long).
Initially I determined the optimum conditions for PCR using a plasmid containing my gene of interest. This worked well using only 10ng of cDNA, quantified using a nanodrop. I then wanted to determine if I could repeat this using cDNA from one of my cancer cell lines, the results are in image 1. The lane order is 350ng, 200ng, 100ng, 50ng, and 10ng of cDNA template. The final lane is the PCR for the plasmid containing my fragment of interest. Surprisingly I the best results were seen with a lot of template, 350ng. So far so good. When I tried to repeat the PCR using 250ng of my breast cancer samples cDNA I got the smears you see in lanes 1-12 in image 2, with the last lane being 350ng of my template cDNA. When I reduce the amount of template I still see the smears.
Obviously my reagents arenít contaminated as the final lane PCR is fine. So next I checked the RNA quality on the nanodrop. The 260/280 ratio and 260/230 ratio were both about 1.95, which should be fine. The cDNA was PCR cleaned after RT and had a 280/260 ratio of 1.85-1.95 and a low yield of 20-30ng/ul from about 1ug of RNA.
So I am now at a loss as to the reason for this smearing, nobody in my department has any idea. I have read that smearing may be the result of too much template or that I could maybe reduce the amount of pfu polymerase, My samples are very limited which make optimisation using the breast cancer samples very difficult so I would like the opinion of people more experienced than myself before I start changing random things. Any suggestions would be greatly appreciated.
Thanks in advance