Frequently Asked Questions About Cell Sorting
Below you will find some of the questions that we are frequently asked concerning cell sorting. If you have a question that is not listed below, please contact us.
Could you please provide advice and recommendations for sorting cells and RNA extraction?
Yes, we have created a suggested guide for sorting and extraction for those who are interested. The guide is intended to serve as starting point for those who may be unfamiliar or uncertain about obtaining the RNA starting material necessary for arm-PCR amplification. However, there are many alternative methods for obtaining cell populations such as FACs and a multitude of methods for RNA extraction/precipitation, which are not discussed in the guide. The guide can be downloaded here, or by going to our document center.
We are planning to sequence a small population of Ag-specific T cells. Can we construct the library with carrier cells instead of carrier RNA like they do in the TCR spectra-typing? And if we can, what kind of cell lines you would recommend as carrier cells, like COS-7?
Currently, we have not done any experiments using carrier cells; therefore, we cannot offer recommendations at this time for using a carrier cell population. If you would like to do this, we highly recommend doing a negative control experiment with the cell population you have chosen as the carrier. In other words, extract the RNA from the carrier cells, and perform a negative control amplification experiment with iRepertoire’s primers. In addition, the optimal amount of total RNA added to the reaction will have to be determined empirically because the amount of RNA from the cell population of interest will not be known. Typically for small cell populations, we recommend that you use the Qiagen RNAeasy Micro kit with carrier RNA. In this case, you would use carrier RNA before extraction and elute in a small volume so that all of the sample RNA can be included in the reaction.
My cells are not sorted; therefore, I have total RNA from whole blood or PBMCs. Should I use more RNA in my reaction?
Yes, we would recommend using more RNA if possible. Please keep in mind that 100 ng is the minimum recommended starting material, but you will likely increase the diversity of the library if you use more input RNA (not exceeding the maximum input of the Qiagen One-Step RT-PCR kit (≤ 1µg per 25 µL reaction)). When RNA from sorted cells is used, we typically find that we can use less starting template. When using total RNA from whole blood or PBMCs, we recommend using the maximum input of the Qiagen One-Step RT-PCR kit.