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Why do I get a low RNA yield from THP-1 / primary monocytes?
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+ Thp-1
+ Rna extraction
+ Rna isolation
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PandaWarrior
Why do I get a low RNA yield from THP-1 / primary monocytes?
my guess in 10s of reading is that you probably don't have enough cells (alive and well) although this may work and my colleague does this by splitting 2 million THP-1 cells, 1/2 for protein and 1/2 for RNA (30-200 ng/uL yields), you may as well save some time and double the cell number and/or find the issue in the technique or kit. We usually use the Aurum Total RNA Mini Kits btw. I find with any RNA isolation, it is always about how much I started with...
my guess in 10s of reading is that you probably don't have enough cells (alive and well) although this may work and my colleague does this by splitting 2 million THP-1 cells, 1/2 for protein and 1/2 for RNA (30-200 ng/uL yields), you may as well save some time and double the cell number and/or find the issue in the technique or kit. We usually use the Aurum Total RNA Mini Kits btw. I find with any RNA isolation, it is always about how much I started with...
I haven't got experience extracting RNA from primary human monocytes, but my experience of working with primary murine macrophages (bone-marrow derived) is that you always get a much lower yield than with macrophage cell lines. This might just be a cell size thing but I'm not sure. My guess based on what you're saying is that it's probably just that you're not using enough primary monocytes. I don't know the kit that you're using, but do you add the lysis buffer directly to the wells? If not, that might be a way to improve yield.
I haven't got experience extracting RNA from primary human monocytes, but my experience of working with primary murine macrophages (bone-marrow derived) is that you always get a much lower yield than with macrophage cell lines. This might just be a cell size thing but I'm not sure. My guess based on what you're saying is that it's probably just that you're not using enough primary monocytes. I don't know the kit that you're using, but do you add the lysis buffer directly to the wells? If not, that might be a way to improve yield.
Thank you Abderrahmane Bengrine, I tried repeating the same protocol today with THP-1 cells, and this time I've got 200 ng/ul which is way enough for qPCR. I think with macrophages, most of them are dead after scrapping, so I'll try a different method to detach them from the wells.
Thank you Abderrahmane Bengrine, I tried repeating the same protocol today with THP-1 cells, and this time I've got 200 ng/ul which is way enough for qPCR. I think with macrophages, most of them are dead after scrapping, so I'll try a different method to detach them from the wells.
Did you verify the viability of the cells after seeding them? Probably the majority of your cells are dead. this is one explanation. Try to extract the RNA with the same protocol from different cells like the one your friend is working on that way you may verify if the problem is with you kit .
Did you verify the viability of the cells after seeding them? Probably the majority of your cells are dead. this is one explanation. Try to extract the RNA with the same protocol from different cells like the one your friend is working on that way you may verify if the problem is with you kit .
my guess in 10s of reading is that you probably don't have enough cells (alive and well) although this may work and my colleague does this by splitting 2 million THP-1 cells, 1/2 for protein and 1/2 for RNA (30-200 ng/uL yields), you may as well save some time and double the cell number and/or find the issue in the technique or kit. We usually use the Aurum Total RNA Mini Kits btw. I find with any RNA isolation, it is always about how much I started with...
my guess in 10s of reading is that you probably don't have enough cells (alive and well) although this may work and my colleague does this by splitting 2 million THP-1 cells, 1/2 for protein and 1/2 for RNA (30-200 ng/uL yields), you may as well save some time and double the cell number and/or find the issue in the technique or kit. We usually use the Aurum Total RNA Mini Kits btw. I find with any RNA isolation, it is always about how much I started with...
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I have scrapped 1X10^6 THP-1 macrophages. How much I can expect the total RNA yield by Trizol method?
I have scrapped 1X10^6 THP-1 macrophages. How much I can expect the total RNA yield by Trizol method?
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I haven't got experience extracting RNA from primary human monocytes, but my experience of working with primary murine macrophages (bone-marrow derived) is that you always get a much lower yield than with macrophage cell lines. This might just be a cell size thing but I'm not sure. My guess based on what you're saying is that it's probably just that you're not using enough primary monocytes. I don't know the kit that you're using, but do you add the lysis buffer directly to the wells? If not, that might be a way to improve yield.
I haven't got experience extracting RNA from primary human monocytes, but my experience of working with primary murine macrophages (bone-marrow derived) is that you always get a much lower yield than with macrophage cell lines. This might just be a cell size thing but I'm not sure. My guess based on what you're saying is that it's probably just that you're not using enough primary monocytes. I don't know the kit that you're using, but do you add the lysis buffer directly to the wells? If not, that might be a way to improve yield.
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Thank you Abderrahmane Bengrine, I tried repeating the same protocol today with THP-1 cells, and this time I've got 200 ng/ul which is way enough for qPCR. I think with macrophages, most of them are dead after scrapping, so I'll try a different method to detach them from the wells.
Thank you Abderrahmane Bengrine, I tried repeating the same protocol today with THP-1 cells, and this time I've got 200 ng/ul which is way enough for qPCR. I think with macrophages, most of them are dead after scrapping, so I'll try a different method to detach them from the wells.
More
VOTE
Did you verify the viability of the cells after seeding them? Probably the majority of your cells are dead. this is one explanation.
Try to extract the RNA with the same protocol from different cells like the one your friend is working on that way you may verify if the problem is with you kit .
Did you verify the viability of the cells after seeding them? Probably the majority of your cells are dead. this is one explanation.
Try to extract the RNA with the same protocol from different cells like the one your friend is working on that way you may verify if the problem is with you kit .
More
VOTE