QuantiTect Primer Assays (for use in real-time RT-PCR with SYBR Green detection) From Qiagen

Polymerase chain reaction (PCR) is used to amplify a fragment of DNA via enzymatic replication across several orders of magnitude, generating millions or more copies of the initial sequence. PCR is commonly used to accomplish a variety of tasks, such as isolation of genetic material, DNA amplification and DNA quantification. If coupled to a reverse transcription (RT) reaction, PCR is also used for quantifying the amount of mRNA expressed in cells. The PCR based method with the highest level of accuracy is quantitative real-time PCR (qPCR). qPCR uses fluorescent dyes to label double-stranded DNA, e.g. SYBR Green or fluorophore-containing DNA probes such as TaqMan® probes, to measure the amount of amplified product in real time.

The use of SYBR Green for the detection of DNA amplification presents many advantages over the use of probes; one key advantage is the price. The main disadvantage of SYBR Green is that the specificity of the reaction is defined by the primers only; therefore, primer design and testing should be particularly accurate, if SYBR Green is used. Qiagen offers pre-designed primers for the detection of any human mouse or rat gene. I use SYBR Green detection for fast and inexpensive experiments, as well as for testing the silencing efficiency after transfection with siRNAs. For studying the expression of “important” genes, I prefer the TaqMan® technology.

The QuantiTect Primer assays are pre-designed, ready-to-use primer pairs. The advantage of buying ready-to-use primers is that the experiment may start immediately without spending time designing and testing primers. However, no information about the primer sequences is provided. Moreover, ready-to-use primers are more expensive than HPLC grade custom oligos; this may be an issue if long-term experiments are planned.

I used QuantiTect primer assay for the detection of rat and human genes, as well as for measuring the amount of 18S rRNA. For some of the primers I used, I tested primer efficiency by amplifying a serial dilution of a pool of samples; all the primers tested were highly efficient (the efficiency was about 2, or a doubling of PCR product with each cycle, which is optimal). Although for long-term experiments, I prefer to design my own primers, I would recommend QuantiTect primer assay for those who wish to use SYBR Green detection without needing to design and test primers.