I have a PRS Santana SE with tremolo. I don't use the tremolo and actually ordered a stoptail version, but ended up receiving a tremolo one. Oh well, i cut down a piece of wood to block the tremolo and act as the stop tail inside the underneath part of the tremolo bridge system.... anyway

For the most part the guitar stays in tune very very well. My problem is the low E string. I use a Boss TU-2 pedal to tune, and the Low E is always in tune plucked open, but when i play say on the 5th fret (an A) the note is noticeably sharp.

I immediately assumed perhaps intonation/saddle length was the problem. But I checked the open string tuning with the 12th harmonic and it was perfect on every string. I have fiddled with the truss rod and saddle height to own liking, but i am not aware of how that could cause this problem....

Any ideas/further questions that might provide an answer? Perhaps i should just take the guitar into a professional? Do Guitar Centers happen to do setup work, and any estimates on cost?

Tuning is never going to be perfect up and down every fret. Musician's generally accept that fact. There's some odd calculation that is used to place each and every fret based on neck scale. It works in theory, but there are a lot of other factors that are not considered (string height, finger pressure, gravitational pull of the moon, wife's mood), which is why they won't be perfect on every fret.

If it's that much of a problem for you though, you could consider a True Temperament neck. I think the Buzz Feitesomethingorotherwhateveryoucallit system does something similar but isn't as spot on as the True Temperment necks are. Most people just live with it really.
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It's neck could be a tad bent.

But it's just natural on a guitar.

There's more than one way to tune to standard (if you are picky.)

If you were going to play Open chords and such, you could just tune the open strings, like normal.

If you were going to be midrange and higher(5th fret and higher.)

Then you would tune with harmonics by matching pitches on other strings.

I haven't done the ladder in ages, but I guess I just never hear the off sound.
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well i was just using the 5th fret as an example. I checked up and down the fret board. its the same the whole length of the string.

Another way to put it. If i tune all 6 strings with the tuner, all of them sound in tune when playing other than the low E. If i tune the Low E down about 1/4 of a whole note, it sounds perfectly in tune, but comes up on the Boss TU-2 2-4 LEDs sharp. I have checked the pedal with other guitars and i am sure it is not the pedal.
in a guitar store they can charge you around 50$ for a full set up.