I am trying to isolate total RNA from FACS sorted CD4 T cells for RNA-Seq purpose. However, I get the low yield of RNA (about 1.5 ug) from 1-2.5 million FACS sorted CD4 T cells. Since I need upto 2 ug of total RNA for miRNA library construction, is it possible to sort CD4 T cells by Miltenyi CD4 MACS beads first and then sort the enriched CD4 T cells by FACS. How can I increase the yield of RNA?
Yes, but would be good to allow for ~24 hour recovery time in culture after FACS sort before MACS sort.
Right now you are hitting the maximum yield of RNA for that number of cells (see the attached PDF from Miltenyi, which is a great little reference). If you want more RNA then you will need more cells. If you are having trouble because you are spending too much money sorting then consider switching tissues (i.e. from spleen to lymph node, where CD4+ T cells make up a much higher proportion). If switching tissues is not possible, then I would do an untouched MACS isolation (kits from Dynal, a division of Thermo). Untouched isolation reduces the risk of your CD4 cells being accidentally activated due to antibody binding. We use these all the time with great success. If you then need really high purity (i.e. >95%) then you could run a very quick sort to further enrich your CD4+ T Cells.
A quick note for the untouched MACS kits, in my experience you get better viability using the isolation buffer they recommend (uses BSA) rather than traditional FACS buffer.
By using MACS MicroBeads for your cell isolation, and then sorting enriched CD4 T cells by FACS, you will get highly pure CD4 T cells, which is good. Regarding your total RNA preparation, don’t load whole cell lysate from 2.5 million cells into one RNA column; this will lead to problems with elution due to overloading. You can increase the yield of total RNA using Trizol or Trizol combined with the RNA columns ( e.g., Qiagen RNeasy kit).
By using MACS microbeads, you will get a good CD4 T cells for RNA seq. you do not need to use FACS; after sorting by MACS microbeads, let the cells to recover from the sorting stress by culture them overnight or for two days, then start with RNA isolation.
Good Luck
Shaima
I tried to sort CD4 T cells using MACS Miltenyi CD4 beads, but I could get only 3 million CD4 T cells. Can anybody suggest how to increase the cell number as I think this number is too low from one spleen.
An 8-week old C57Bl/6 mouse spleen is about 20% CD4+ cells, while peripheral LN are about 40%. In my experience, processing an entire spleen from an animal at 8 weeks you would get you about 19 million CD4+ cells. Processing total LN (which would save you a lot of MACS reagent) would get you about 14 million CD4+ cells. Are you using mice in the 6-10 week age range? How many live cells are you getting from the spleen (just do a trypan blue stain and count on a hemocytometer)? Are you using enough antibody for your MACS selection? Processing roughly 100 million cells (one spleen) requires a lot of antibody.
Hi Erik,
I used 7 weeks old mouse. I counted the viability of cells using trypan blue. I can get usually 50-60 million cells from one spleen. Very rarely, I get up to 70 million cells. I incubated the cells from one spleen with 10 ul CD4 MACS beads. That means at some step I am losing cells. Please suggest.
Best,
Tabasum
Tabasum,
Those numbers look good for a mouse at that age, 8 week old animals have more splenocytes due to B cells finishing their peripheral development around 8 weeks and filling the niche. I think the issue is with the amount of antibody you are using. Looking at the product sheet for Miltenyi's MACS (L3T4) beads they use 10 uL for every 10 million cells. So you were getting about 30% of the 10 million worth you had antibody for, which is about right. The protocol says you should be using 50-60 uL of the beads. I think this will solve your numbers issue.
Hope this helps. Good luck.
I would definitely suggest running analytical FACS on your MACS-isolated CD4+ T-cells to see the % of CD4+ T-cells as a measure of purity, if you plan to use them for downstream applications. If you plan to use FACS sorting after MACS this is not necessary, but then if you have enough flow time you can just FACS sort directly without using MACS.
While the MACS method works well with standard spleens, various factors like infection or unusual mouse strain can significantly lower the purity after isolation because the non-CD4+ populations are not accounted for by the kit specs. Since this is a negative selection procedure, any cells which are not captured by the kit can end up in your prep. I have seen a purity as low as 60% in some infected spleens after MACS...
Purifying CD4 positive T cells from mouse spleen using the CD4 positive T cell isolation kit (Miltenyi Biotec) and autoMACS Pro separator, followed by cell sorting, is a good approach. I recently was successful in obtaining purified CD4 positive T cells using this approach. In my experiments, I obtained 86-91% of CD4-positive T cells by the negative selection procedure. PE anti-mouse CD4 antibody (eBioscience) is good for FACS. I suggest you use the isolated spleen cells prior to purification to practice total RNA isolation, using TRIzol combined with the columns from the QIAGEN RNeasy Mini kit to determine how many cells you need to use to obtain 2 micrograms of RNA. With good skills for isolating total RNA from spleen cells, you start to use your purified CD4 positive cells. In addition, the isolated spleen cells should not contain more than 20% dead cells.
Hope this is helpful.