real time PCR

[bharuch]

Hi! everyone,
We perform quantitative PCR on BCR/ABL( oncology) on blood and bonemarrow specimens. BCR/ABL is reciprocol translocation of one gene from chromosome 9 to chromosome 22)
our fusion gene( BCR-ABL) is inserted into plasmid.
then we dilute plasmid from 1000000 copies to 10 copies. We run real time PCR to develop our standard curve.
for 1000000 copies Ct= 21.5
10000 Ct = 27.0
1000 Ct = 30
100 Ct= 33.5
10 Ct= 36.5 ( lowest dilution on our standard curve)

our sensitivity of the assay is 10 copies for fusion gene( BCR/ABL)

similarly we insert our control gene(ABL) into plasmid and we prepare standard curve of control gene too.

when we run our samples we run calibrators plasmid ( fusion gene with 1000 copies so cutooff Ct= 30 and control gene with 1000 copies so Ct= 30.0).
We get Ct value on patient on both fusion gene( BCR/ABL) and control gene( ABL)
we export both result onto our software to get normalized ratio in exponential format.

my question is if the instrument is giving us reading Ct = 38.5 can we export this date to get final report . In other words can we give results below out sentivity level( 10 copies which is CT= 36.5)
sometimes our blank on both fusion gene and control gene gives reading 39.0 what does it indicates.
Before we had a cut off value of 36.0 that is equivalent to 10 copies of fusion gene. Now we decided not to have a cut off value and gives result if we get reading such as 38.0 or 39.0 and get normalized ratio( BCR-ABL/ABL) in exponential format.

If we use protocol telling us that prepare calibration curve using plasmid( with inserted fusuon gene) from 1000000 to 10 copies( Ct 21.5 to 36.5) then it is ok to give value above 36.5 I mean until we do not get any reading from instrument( lightcycler).


Thanking you