RT-PCR works but not consistent - (Nov/16/2008 )
I current have some problem of inconsistency.
There are at least 6 pairs of primer set to amplify ONE gene of interest (at difference position of RNA).
For the most of cell lines, all of primer sets work fine; however, there are 2 strange cell lines can only be amplified by 1 particular primer set not the others. I have repeated 2 times and get same result.
Western Blot confirms that there is protein in those cell lines, and sequencing confirms PCR product is our gene.
How is that possible?
5 of 6 primer set fail to detect the existent of cDNA in these 2 cell lines, but work fine in other cell lines?
And there is 1 more strange cell line (I think it done NOT like me). Because when I grow them, I cannot detect the expression of this gene using all 6 primer pairs; when other person grow it, however, any of the 6 primer pair can amplify the fragment of this gene (at very low level but detectable).
In latter trials, I am the only person doing RT and subsequent PCR.
As I can see, both of us use the same medium and incubator. She taught me the handling of cell culture, and both of us agree that we have similar technique of handling.
The difference I notice is when we decide to harvest the cells. I harvest the cell at more confluent condition. I also try harvest at the confluent level she used and still got nothing.
Can anyone give some suggestion about what happen here?
Is there any way I can do to deal the problem of inconsistency?
Great thanks for any suggestion.
Why the heck do you need so many primers for the same gene?
Just get you colleague to harvest. mRNA must like her better.
Just get you colleague to harvest. mRNA must like her better.
There are several alternative splicing variants in this gene, 4 of 6 primer is to detect the level and ratio of variants.
Probably I am too ugly, the cells and RNA don't like me
That is our head post-doc said: dependent on hypothesis, if we don't want the the gene come out I should do the work, and if we need it come out she will do the culture
haha
that's funny, bro.
i guess her decision of confluency influences you results so much the gene expression must depend on the stage of confluency. perhaps she can decide when to harvest then you do the work, mayb one flask split 2 and you 2 do at same time. Then compare the results.
good luck