Hi guys,
I haven't had this happen to me in a few years: I used Sera-Mag Oligo-dT beads with homemade buffers to enrich polyA RNA from high-quality RNA, and got nuttin'. I'm trying to come to terms with perhaps needing to go back to the extraction phase, with one sample that I can't replace without significant delay. While I get through the grief phases, I have some questions for you all.
Background: I went through a practice run last week to make sure I could still do this, and retrieved 1.2% and .9% polyA from my total RNA samples. That's good! It's what I expect from those samples.
This week, I took 16 of my 48 samples (in random order, you stay away, batch effects!) and took them through the same exact procedure. I quantified with Qubit RNA HS and all of my yields were below quantitative concentration. Even when I kicked up the input amount to 5 uL, it was still as though I was just quantifying water.
I then quantified the supernatant that I saved from the first binding step. It has a little more RNA in it than I expected from my samples, but I did add RNA spike-ins, and that probably accounts for the bit of extra.
The only things that were different this time from last time:
- I may not have washed all the Sodium azide off the beads as completely as I usually do. Will Sodium azide prevent polyA capture?
- I added RNA Spike-ins, which I've never done before. But these are just RNA in pure water. Surely that didn't affect anything?
- I used a different lot of beads. I really want to believe that the new beads are bad somehow, and that all of my beautiful mRNA is still in my supernatant tubes, and that my hoarding of waste products is a good practice and not the sign of a demented paranoid mind.
I'm tempted to use the "old" beads with a couple of samples of supernatant, and see if I get anything back. That'll be about three hours of work. I could A/B test, I guess, with old and new beads, but I'm worried that that means I'll lose half of my samples. I don't have a lot to spare.
In the meantime, I guess I'll go collect some new specimens for the one I can replace. Why can't things just go smooth?
I haven't had this happen to me in a few years: I used Sera-Mag Oligo-dT beads with homemade buffers to enrich polyA RNA from high-quality RNA, and got nuttin'. I'm trying to come to terms with perhaps needing to go back to the extraction phase, with one sample that I can't replace without significant delay. While I get through the grief phases, I have some questions for you all.
Background: I went through a practice run last week to make sure I could still do this, and retrieved 1.2% and .9% polyA from my total RNA samples. That's good! It's what I expect from those samples.
This week, I took 16 of my 48 samples (in random order, you stay away, batch effects!) and took them through the same exact procedure. I quantified with Qubit RNA HS and all of my yields were below quantitative concentration. Even when I kicked up the input amount to 5 uL, it was still as though I was just quantifying water.
I then quantified the supernatant that I saved from the first binding step. It has a little more RNA in it than I expected from my samples, but I did add RNA spike-ins, and that probably accounts for the bit of extra.
The only things that were different this time from last time:
- I may not have washed all the Sodium azide off the beads as completely as I usually do. Will Sodium azide prevent polyA capture?
- I added RNA Spike-ins, which I've never done before. But these are just RNA in pure water. Surely that didn't affect anything?
- I used a different lot of beads. I really want to believe that the new beads are bad somehow, and that all of my beautiful mRNA is still in my supernatant tubes, and that my hoarding of waste products is a good practice and not the sign of a demented paranoid mind.
I'm tempted to use the "old" beads with a couple of samples of supernatant, and see if I get anything back. That'll be about three hours of work. I could A/B test, I guess, with old and new beads, but I'm worried that that means I'll lose half of my samples. I don't have a lot to spare.
In the meantime, I guess I'll go collect some new specimens for the one I can replace. Why can't things just go smooth?