Q: What information do I need to provide to design the primers and probes?
A: Please email the NCBI accession number or the coding sequence for the target(s) you are interested in. Please note if there is a specific region you wish to study.
Q: What software program does the Core use to design the primers?
A: The Core uses Primer Express™ 1.5 from Applied Biosystems to design all primers and probes.
Q: What different detection systems does the Core provide?
A: We currently use the classic TaqMan system which contains the forward and reverse primers plus a specific dual-labeled probe. The TaqMan probes can be used for both Real-Time qPCR as well as digital PCR. We also use SYBR Green.
Q: Which detection system is better (TaqMan or SYBR Green)?
A: Both systems work very well. Factors such as sample concentration, the number of samples, and the number of targets you wish to study will decide which is better for your needs.
Q: What fluorescent dyes do you use for the probes and which can be multiplexed together?
A: Most reporter dyes are 6-FAM and TET with a Black Hole Quencher and these can be multiplexed together. Other dyes that can be used are VIC and JOE.
Q: What reference genes (normalizers) does the Core provide?
A: We currently have human, mouse and rat GAPDH, human and mouse Beta Actin, and ribosomal 18S that recognizes human, mouse, and rat.
Q: What reference genes work better with certain samples?
A: Reference genes can be influenced by the experimental situation. To see what is best for your project we suggest reading papers that are similar to your studies. 18S seems to work better across samples from different cell lines and cell cultures as GAPDH can be affected by cells in different stages. It also seems to work better for tissue samples.