QPCR validation of gene fusion on clinical samples

由 sufang 在 二, 04/08/2014 - 09:42 發表 Cholangiocarcinoma Dan Robinson MCTP RNA-seq UMich Yi-Mi

For QPCR validations we use real-time PCR with SYBR green on an ABI instrument such as a StepOne, we set the run for 40 cycles, cDNA from 200 ng or so of toral RNA. Using GAPDH control primers we get a Ct of 18 cycles or so.

For a real fusion, the Ct can be anywhere from 24 to 33 but not much above that. We use ABI Fast SYBR Green mastermix kits with UNG for our PCRs. One trouble with the PCR approach is the number of 3’ partners and the number of possible fusion junctions for each 3’ partner. (The FGFR fusion exon is nearly always the same.)

For QPCR validations we use real-time PCR with SYBR green on an ABI instrument such as a StepOne, we set the run for 40 cycles, cDNA from 200 ng or so of toral RNA. Using GAPDH control primers we get a Ct of 18 cycles or so.

For a real fusion, the Ct can be anywhere from 24 to 33 but not much above that. We use ABI Fast SYBR Green mastermix kits with UNG for our PCRs. One trouble with the PCR approach is the number of 3’ partners and the number of possible fusion junctions for each 3’ partner. (The FGFR fusion exon is nearly always the same.)