Different RNA sources, Different PCR optimal condition? - (Apr/18/2006 )
I try RT-PCR with positive control to find optimal condition for one primer (such as annealing temp, cDNA etc.) but when I used cell from other sources I can not use the same condition with positive control cells.( I use the same condition with positive control cell)
So I must try new optimal condition to every new cell sources?
I don't know why? (same primer, same reagent, same thermocycler)
Do you have any ideas? Please help me.
possibly an obvious question, so excuse me, but your positive is a true positive control yeh? ie. it is expressed in all your target cell types?
I don't see why you would need different conditions, the secondary RNA structure and sequence should be the same for all cell types. Have you quantified your RNA and looked at the quality from the different RNA extractions? Maybe it's a problem of detection limits because the RNA might not be expressed at the same high levels in different cell types.
I'm with Vairus...I was thinking it sounds like an issue of a large difference in template dosage
Thank you very much for your opinions.
My positive control cell is the true positive, and express in my all cell type. (It produced protein detected by other method, so I try to check the gene expression by RT-PCR, but I have problem like the topic.)
The ratio of RNA 260/280 that have problem is about 3.6, Maybe it's cause the problem?
if this is the problem, how can I fix it?
but I treated DNase, before use 1 microgram to convet to cDNA.
and check the quality by GAPDH, it has lighter band than positive control cell band (in the same loading volume.)
If the problem is about expression level how can I solve this problem?
Do you have any ideas, help me please. Thanks again for all your opinions.
wow, a 3.6 ratio could certainly contribute to your problem!
ideally you want 1.8-2.1
high ratios usually indicate contamination by organics/solvents (phenol, alcohols, etc); low ratios usually indicate contamination by protein. both will screw up your results, because you do not have the concentration you think you have...if you have enough sample, I would suggest possibly re-extracting and definitely EtOH (or other alcohol) precipitating, with a 70% wash...then making sure your pellet is dry before resuspending, and running it through the ol' spec again