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Hey all,
I am in search for some help. We have been using the Qiagen RNEasy kits for several years now without an issue. This past January, we had ordered new kits and since then we have not been able to get much RNA off the column despite repeating protocols from previous successful runs. To give you a little background:
We culture KGN treated cells and collect via Trypsinizing. We count the cells, approximately 1-1.5 million per well, and then spin down. We remove ALL of the media, then proceed to process for RNA using the Qiagen RNEasy kit with QIA Shredder step to homogenize.
We do all the steps, inclusive of the DNAse treatment AND optional second spin after second RPE wash. We then elute with 40ul of RNA free water (no incubation), and after that spin, re-apply the eluate back onto the column for a second spin. Our yields have typically (and consistently) been 200-400ng/ul in the past for cell counts ranging from 750,000 to 1.5 million.
As of late, we are lucky to get 40ng/ul - same treatment, same protocol, same steps with Qiagen. We have been back and forth with Qiagen, they have sent multiple test kits, in all instances, we are lucky to get 40ng/ul.
To alleviate dealing with cells, I made up a stock of pure RNA, Nano reading show purity at a concentration of 900ng/ul. I seed 36ul of that into a tube, add lysis buffer, and what I found was that the RNA is not coming off the column. In all steps, I show nothing coming through BUT the RPE and RW1 buffer coming through the column. 260/280 readings in spectrophotometer show zero RNA on those steps. So we go to elute - nothing comes off the columns all of a sudden.
Qiagen is stumped - so are we! Any and all ideas at this point are welcomed as we are desperate to figure out what the culprit is.
Thanks in advance!
I am in search for some help. We have been using the Qiagen RNEasy kits for several years now without an issue. This past January, we had ordered new kits and since then we have not been able to get much RNA off the column despite repeating protocols from previous successful runs. To give you a little background:
We culture KGN treated cells and collect via Trypsinizing. We count the cells, approximately 1-1.5 million per well, and then spin down. We remove ALL of the media, then proceed to process for RNA using the Qiagen RNEasy kit with QIA Shredder step to homogenize.
We do all the steps, inclusive of the DNAse treatment AND optional second spin after second RPE wash. We then elute with 40ul of RNA free water (no incubation), and after that spin, re-apply the eluate back onto the column for a second spin. Our yields have typically (and consistently) been 200-400ng/ul in the past for cell counts ranging from 750,000 to 1.5 million.
As of late, we are lucky to get 40ng/ul - same treatment, same protocol, same steps with Qiagen. We have been back and forth with Qiagen, they have sent multiple test kits, in all instances, we are lucky to get 40ng/ul.
To alleviate dealing with cells, I made up a stock of pure RNA, Nano reading show purity at a concentration of 900ng/ul. I seed 36ul of that into a tube, add lysis buffer, and what I found was that the RNA is not coming off the column. In all steps, I show nothing coming through BUT the RPE and RW1 buffer coming through the column. 260/280 readings in spectrophotometer show zero RNA on those steps. So we go to elute - nothing comes off the columns all of a sudden.
Qiagen is stumped - so are we! Any and all ideas at this point are welcomed as we are desperate to figure out what the culprit is.
Thanks in advance!