Hey,
So I have question regarding total RNA extracted from CD14+ cells in comparison to THP-1s. When working with approximately 2 million CD14+ cells, I typically end up with about 1-1.5 (often less) ug total RNA relative to about 10-12 ug in THP-1s of the same number. My A260/A280 ratios range from 1.9-2.2.
CD14+ Cell Isolation: CD14+ cells are positively selected (MACS Beads), rested for 2-12 hours and then stimulated for 24 hours.
Cell Culture: Cells are incubated in a 24 well plate with any adherent cells being detached, washed and lysed along with those in suspension. Trypan staining shows that viability is high post stimulation.
Extraction method: I have been using Qiagens RNeasy Plus extraction kit as per the manufacturer's protocol (including all optional steps). I have also tried using TRIzol but I see the same thing happening. This has been problematic as I am looking to analyse low copy transcripts via qPCR which already come out reasonably late in THP-1s.
Has anyone ever faced a problem similar to this? Are these yields normal for patient samples? The number of cells I am using seems like more than enough for my assay but I feel that the extracted RNA yield is the main aspect holding me back, and I have often had to turn to RNA concentration protocols to use my preparations at an appropriate volume for DNAse and RT.
Thanks in advance,
N
Hi Nathan,
In my experience, this is a normal yield for that number of cells. You may see better results using a manual Trizol method of extraction, but that seems to depend on your conditions. Another step that you could improve is your centrifuge step. We have found that slightly altering the speed and time of the centrifuge can often yield more reliable amounts of RNA. Good luck with your research.
It sounds like a normal yield to me. The CD14+ monocytes tend to have lower RNA yields than the CD16+ monocytes. The only thing you need to do here is to concentrate your RNA more. A 500ng/1ug input for cDNA synthesis should be more than enough for qPCR. Also it might be an idea to check your RNA quality and yield on the bioanalyzer since this is much more accurate than the nanodrop. Good luck !!!
Hi all.. we have recently began purifying RNA from healthy donor monocytes using the same qiagen RNeasy Plus mini kit and get about the same yields of 1-2ug. With PBMCs we did not have this problem but with monocytes I noticed our RIN numbers were only 5-6.5 sometimes. I read that monocytes do have intrinsic RNases that could affect RNA quality but I also left our samples frozen in RLT plus lysis buffer at -80 for a few days up to a week.
So my question was whether anyone tried other RNA kits for monocytes or froze down the samples for a shorter period of time? We eventually need to culture the cells for a 5-6hr time point so isolating RNA fresh would really not be ideal if do not get fresh monocytes to stimulate until around 2-3pm. Looking for any tips to increase the monocyte RIN as we used everything fresh (kit, water, ethanol, etc) such that RNase contamination should not have been a huge issue. Does the RLT plus lysis buffer with Beta mercaptoethanol maybe not efficiently inhibit the RNases? I had done these monocyte RNA isolations a few years ago without any issues so now not sure why the RINs are lower... Thanks
I first do a PBMC isolation and then I FACS sort the three monocyte subsets straight into Trizol-LS and isolate RNA immediately after with the Zymogen DirectZol kit and froze down in -80. I typically get high RIN numbers with 8 being the lowest. RNA yields for the classical CD14+CD16- (500,000 cells) are between 0.8-1ug and the intermediate CD14+CD16- (80,000) are only around 0.2ug. These values were measured after the RNA was in -80 for 6 months.
Thanks Anouk! I had never heard of that directzol kit and knew that trizol would give higher yields so would be curious to know how much RNA you get from 1million or 0.5million monocytes using this method. I will definitely get a free samples if I can. Would be fast enough to do right away or freezing in trizol may be more stable versus RLT lysis buffer.
Good day, we have a problem with RNA yield and quality after FACS sorting.
We try to isolate total RNA from blood of healthy patient and breast cancer patient for RNAseq. We FACS sort the three monocyte subsets (СD14+16- - 100,000-500,000, CD14+16+ - 5,000 - 15,000, CD14+16- 15,000 - 50,000) straight into RLT buffer + beta mercaptoethanol and isolate RNA immediately after with the Qagen Micro kit (40-60 min after sorting). Unfortunately we can't get a permanent RNA yield and quality. In the first day we have RIN 9,5 and 42ng from 100K (14+16-), but in the next day we have RIN 2,4 and 5ng from 500K(14+16-) from the same donor. We prepere cells to sort in laminar flow cabinet, FACS sort without laminar and isolate RNA in other laminar flow cabinet (only for RNA isolation). Did you have such problems? Can you suggest any advices to to solve it? Thanks in advance,
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