Frequently Asked Questions
Q: I would like to compare the expression levels of two genes in each of our cell lines, what do I need to do that?
A: You need to design primers and probes for those genes. In addition, you need an endogenous control gene, or housekeeping gene, which is expressed in a stable level in all the cell lines. The results of the genes of interest will be calibrated against the housekeeping gene. This will enable a relative comparison of expression between the cell lines.
Q: What’s the idea in using a probe in a TaqMan assay?
A: The probe is used for quantifying the amount of gene of interest in your sample. A PCR isn’t quantifiable without some method of measuring the accumulation of the PCR product, and with a TaqMan probe, the PCR product can be quantified in real time as the reaction proceeds. The probe attaches to a sequence-specific location in the amplified DNA strand, is cleaved by the polymerase enzyme and emits light, which is detected by the instrument.
Q: What about the results?
A: You will have the results as an .eds document. In the .eds document you can also look at the raw data, for example the amplification plots.
Q: What do the Ct values mean?
A: The Ct value means the number of PCR cycles where the reporter dye signal is sufficiently high to cross an automatically or manually determined threshold value. The earlier the signal passes the threshold value, the higher is the expression of that gene in that sample. In a standard setup 40 cycles are run. Ct “undetermined” means that the signal stays under the threshold during all the 40 cycles, which practically means a negative result, or that the expression is so low that it can’t be detected.
Q: What do the delta Ct values mean?
A: In sample handling, small differences between samples can occur. To minimize the effect of these differences, all results are normalized against an endogenous control, or housekeeping gene. Delta Ct is calculated by subtracting the Ct of the housekeeping gene from the Ct of the gene of interest.
Q: How is the difference in expression levels calculated?
A: By comparing the delta Ct:s, you can calculate delta delta Ct:s by subtracting one delta Ct from the other. The delta delta Ct is used to calculate the relative difference in the expression levels between samples. For example, if the Ct:s of sample x and sample y are 29 and 26, and the corresponding housekeeping gene Ct:s are 24 and 23, respectively, the delta Ct:s are 5 for sample x and 3 for sample y, and the delta delta Ct is 5-3=2. The difference between samples is 2 to the power of delta delta Ct, in this case 2 to the power of 2, which means that sample y is expressed 4 times more than sample x.
Q: How does your Full Service work?
A: The full service includes quantitative real-time PCR and reviewing the results. We run 4 replicates of each reaction, plus no template controls, so for example analyzing four samples means 20 reactions/gene and five samples 24 reactions/gene. The full service price includes PCR plastic materials, master mix and working hours needed for pipetting and reviewing the results. Customers can choose to make cDNA and design and order primers and probes themselves. If we prepare the cDNA and/or design and order the primers and probes, those costs will be invoiced in addition to the full service price.
Q: How to choose primers and probes?
A: You have several options:
• TaqMan® Gene Expression Assays – Optimized, ready-to-use TaqMan reagent based assays. For information on available primer/probe sets or to place an order, go to:
• Custom TaqMan® Gene Expression Assays – Design, synthesize, formulate, and deliver quality-controlled primer and probe sets. Use this service if the assay you need is not currently available.
• design primers and probes using the Applied Biosystems Primer Express® Software and order them from any oligo supplier.
• choose your probes from Roche Universal Probe Library using their assay design center. The probes are available through us.
Q: Which TaqMan instruments do you have and how can I use them?
A: We now have one machine, QuantStudio 12K Flex Real-Time PCR System. With that we can run 96-well and 384-well plates. The instrument is operated by our staff: you can bring us a plate ready to be run and the associated EDS document or excel sheet, and we run the plate for you.
Q: Where can I find the reagents and materials needed for the PCR?
A: We have a stock of plates and seals in our common plastic ware storage. If you would like to get ProbeLibrary probes, send an e-mail to taqman (at) utu.fi and the probes will be taken to the “Kyllikki” freezer room in BTK (5th floor, room number 5074) in three working days.
Q: Should I use SYBR Green or TaqMan probe chemistry or ProbeLibrary probes?
A: All have their advantages. SYBR Green is cheaper, but it often needs more optimization for each gene. So if you have time and only few genes, it’s an option. The TaqMan probes usually work right away with a standard setup without the need of optimization. For a small amount of samples the ProbeLibrary probes are cheaper than longer TaqMan probes and usually work without optimization like the TaqMan probes.