Apologies if this doubles. I tried to post before but it didn't seem to work.
I've had very good success with poly(A) / mRNA enrichment on Sera-Mag Oligo-dt beads in the past. In fact, I had great success just last week. This week, it seemed to fail. I'm trying to figure out why, and if there's any hope I might rescue my samples.
I use home-made buffers with oligo-dt beads from Sera-Mag. This time (as opposed to last time) I also used RNA Spike-Ins from ERCC. Upon Qubiting my eluted "mRNA" this morning, I determined that I recovered essentially no polyA RNA.
Differences this time from last time:
- I opened a new lot of beads. Is it possible the beads are bad? (I'm really hoping for this.)
- It is possible I didn't wash off all of the Sodium azide solution, I was multi-channeling and having trouble seeing what I was doing. Would sodium azide inhibit polyA capture?
- I used RNA Spike-Ins, but I did not expect this to cause a problem. It's just a little RNA in molecular-pure water.
I Qubited the supernatant that I'd saved from the first wash (should be mostly rRNA in 1x Binding Buffer) and it actually had a little bit more RNA than I'd originally had in my sample. Possibly the additional RNA spike-in could account for there being slightly more RNA.
Should I try redoing this with the supernatant from a couple of the samples, using the "old" beads I used last week that were fine? Should I consider A/B testing these new beads?
Thanks for any advice you can provide.
I've had very good success with poly(A) / mRNA enrichment on Sera-Mag Oligo-dt beads in the past. In fact, I had great success just last week. This week, it seemed to fail. I'm trying to figure out why, and if there's any hope I might rescue my samples.
I use home-made buffers with oligo-dt beads from Sera-Mag. This time (as opposed to last time) I also used RNA Spike-Ins from ERCC. Upon Qubiting my eluted "mRNA" this morning, I determined that I recovered essentially no polyA RNA.
Differences this time from last time:
- I opened a new lot of beads. Is it possible the beads are bad? (I'm really hoping for this.)
- It is possible I didn't wash off all of the Sodium azide solution, I was multi-channeling and having trouble seeing what I was doing. Would sodium azide inhibit polyA capture?
- I used RNA Spike-Ins, but I did not expect this to cause a problem. It's just a little RNA in molecular-pure water.
I Qubited the supernatant that I'd saved from the first wash (should be mostly rRNA in 1x Binding Buffer) and it actually had a little bit more RNA than I'd originally had in my sample. Possibly the additional RNA spike-in could account for there being slightly more RNA.
Should I try redoing this with the supernatant from a couple of the samples, using the "old" beads I used last week that were fine? Should I consider A/B testing these new beads?
Thanks for any advice you can provide.