Can I multiplex my assays if my amplicons are not all the same length?
Yes, it is possible. The amplicons do not have to be the same length in order to duplex the assays. You need to use different reporter dyes on the probes in multiplex - they should have a substantial difference in maximum emission wavelength to be effective. A classic example is to use FAM and VIC for the 2 probes. Depending on the expression of the targets, you may also need to limit the primer concentration for one of the assays. This is common when duplexing a target and endogenous control (the control probe is VIC labeled and the primer concentration is decreased so the target assay can properly compete for use of enzyme and dNTPs).
I want to order a TaqMan Gene Expression Assay that you have discontinued. Is there a way do that?
Yes, you can order it as a custom assay. Since the assay is discontinued, it can not be ordered on-line. You will have to call or email the order to genomics@appliedbiosystems.com, and include the assay ID. The assay will be synthesized once the order is received. It will be designed and priced as a Custom TaqMan Gene Expression Assay so you will need to choose the scale.
I am getting the same amplification in my no-RT controls as in my samples. What does this mean?
It means that you are probably detecting genomic DNA (gDNA) in your sample. It could be the method you are using to prepare your RNA. Our website has information and products to help you eliminate genomic DNA contamination. Also, if you are using an assay ID with the suffix _s, it will, by definition, detect gDNA. Assays ending with the suffix _s are designed within a single exon. If there is gDNA present in the sample, you must treat with DNase.
Can I use TaqMan Gene Expression Assays for a gene copy number study?
TaqMan Gene Expression Assays with “_s1” as assay ID suffix are the assays whose primers and probes are designed within a single exon, and therefore, will detect genomic DNA. However, the application of these assays in gene copy number studies has not been validated. Other gene expression assays will not detect gDNA, let alone the region of variance.
I am working on both mouse and human samples for gene expression studies. Do you have one endogenous control that I can use with both species?
The 18S rRNA is highly conserved among eukaryotes. We have several configurations of assays for this gene: P/N 4331182, Assay ID: Hs99999901_s1; PN 4319413E, 4310893E, 4333760T, 4333760F, 4308329. You can use any of these for both human and mouse samples, as well as samples from any other eukaryotic species.