Frequently Asked Questions About Primer Orders
Below you will find some of the questions that we are frequently asked concerning primer orders. If you have a question that is not listed below, please contact us.
What is the difference between the V-J and V-C primer systems?
The V-J primers amplify about 100 bp sequences around the CDR3 region and are suitable for both genomic DNA and RNA samples; the V-C primers amplify about 150 bp (Illumina) or 250 bp (Illumina MiSeq & Roche 454) sequences around the CDR3 region and should be used with RNA samples only because there is a large intron in between the C and rearranged V(D)J genes.
Are the primers compatible with gDNA template?
Only our V-J primer systems are compatible with gDNA due to an intron between the rearranged V(D)J and C genes; however, all of our primers are compatible with RNA.
Does the amplified region contain the CDR3 region?
Yes, our primers for human amplification and sequencing for Illumina MiSeq and Roche 454 are located within FR1 and C genes, so they amplify almost the entire antibody sequence including CDR1, CDR2 and CDR3. Our primers for mouse amplification and sequencing for Illumina MiSeq and Roche 454 are located within FR2 and the C gene, amplifying the CDR2 and CDR3. With Illumina, we offer both V-J and V-C amplification primers. The V-J primers amplify about 100 bp sequences around the CDR3 region and are suitable for both genomic DNA and RNA samples; the V-C primers amplify about 150 bp sequences around the CDR3 region and should be used only with RNA samples only because there is a large intron in between the C and rearranged V(D)J genes. With the Illumina primers, both V-J and V-C primer systems may include a portion of the FR3 region in addition to the CDR3 region. The V-C primers will also include the beginning portion of the C-region.
When I order a primer reagent system, do I actually get the primer sequences?
No, you do not get the primer sequences. We use a patented arm-PCR technology (amplicon rescued multiplex PCR), and the primer sequences are not disclosed. You will see the inside primer sequence after sequencing is completed, but the outside primer sequences are not disclosed.
Do you offer primer sets for paired analysis of TCR alpha/beta chain in the single cell level?
Currently, we do not offer this as a product; however, it is in our development pipeline. Please check back later.
How much template RNA do I need for amplification?
For service orders, we require 500 ng of RNA from isolated cells (with an A260/280 ≥1.8) at a minimum concentration of 15 ng/µL for service orders. For PBMCs, total RNA from whole blood, and total RNA from tissue samples, we require 1 µg of RNA at a minimum concentration of 35 ng/µL. For service samples, we request more than is required for a single reaction so that there is enough for a repeat amplification, should it be necessary.
The amount of RNA input is dependent on the starting sample type. If you are using total RNA from whole blood, with no downstream cell sorting, then we recommend using 1 ug of RNA. This is the same recommendation for tumor or various tissue types. The reason for this is that there is a lot of non-immune specific RNA in the sample that is acting as background and you want to make sure that you have enough immune specific RNA going into the reaction. If you are using PBMCs, then you can put less RNA template. We, typically, use 300 - 500 ng for this sample type.
When using cells that have been sorted, you can use even less RNA template because the concentration of immune-specific RNA is greater. When working with sorted cells, we have used as little as 50 ng but it was of good quality and was intact. The important thing to remember when deciding how much RNA template to use, is to think of your sample type and the quality of your RNA. You can also consider that if you are using RNA extracted from a tissue type that has a lot of immune-specific cells, such as lymphoid tissue, then you can get away with using less RNA template in your reaction. The other consideration is if you are comparing one sample to another, then you might want to keep the starting template amount (in ng) the same from both samples.
What also needs to be considered is that the total volume that can be added to the first PCR reaction is 13.75 ul. The RNA needs to be at a concentration that will allow you to put the desired starting template amount (in ng) into this 13.75 ul. If you have a really concentrated RNA sample, then you can make up the difference of that 13.75 ul with nuclease-free water. For example, your RNA sample is 250 ng/ul and want to add 500 ng of template. You would add 2 ul of RNA template and 11.75 of water. This dilution would give you 500 ng of RNA going into your first PCR reaction.
Basically, the more purified your sample type, the less amount, in ng, of RNA template is required to achieve the desired immune-specific amplification.