I am using the PAXgene blood miRNA Kit (Qiagen) to isolate RNA from PAX blood tubes. The tubes were stored at -80 for 6 months, and are thawed for 3 hours before RNA isolation (allowing equilibration to room temperature). The first step in the protocol is centrifugation of the PAX blood tubes at 3000 g for 10 minutes. However, after adding RNAse free water I have trouble dissolve the pellet properly, which I suspect reduces the yield. I have tried to vortex for up to two minutes (e.g 6x20 seconds), but I still get relatively large pieces of pellet that wont dissolve. The same problem occurs after the centrifugation of step 4.
Does anyone have a solution to this?
Thank you for your answer. I will try this the next time.
Is it best to use a water bath?
Can I heat the pellet after adding RNAse free water and buffer (BM2)? Or is it best to just heat the pellet first, and then add the solutions and vortex after?
I've had same problem, and suspected blood clots in sample (see this description : http://www.esciencecentral.org/ebooks/genomics-clinical-trial-assay-development-issues-and-lessons-learned/making-a-gold-standard-assay-even-better.php )
So wondering if you tried the heating, and did it work?
Hi David. I tried heating some test-samples in water bath (55C) for 3 minutes, but samples with a very firm pellet did not dissolve properly. I have not used water bath after this.
However, this autumn we performed a new experiment (exact same design) and I have extracted RNA from over 100 PAX samples. Surprisingly, I did not have the same problem this time. I have tried to figure out why, but have not found the exact reason. I did 3 things different this time (that might be a part of the reason?):
I also ensured that samples were inverted properly both before storage, and after thawing. I hope this helps, although I can not give you an exact answer.
Before my last experiment I also contacted Qiagen and got the following tip:
The PAXgene Blood RNA tubes and Kit are designed and optimized for human blood and we have no data as how it performs with blood from animals. The difficulties with dissolving the pellet may indeed come from the fact that viscosity and cell counts are different for goat and human blood. To dissolve the pellet you can try to introduce the following changes to the protocol:
Additionally, Incubation of the PAXgene Blood RNA Tube (BRT) overnight at room temperature may increase yields.
I never did this, but might be worth a try...
Tusen takk for dette svaret!
I'll try overnight out of the freezer (my samples are -80 for >6 months), lower centrifuge G, and more inversions after thawing. If that doesn't work I'll try the earlier PK as suggested by Qiagen.
Sometimes I get a hard pellet that won't dissolve (~1 in 8 samples) and sometimes a soft, glutinous pellet which won't break up even with pipetting (~1 in 12 samples). I'll let you know how I get on.
Thanks again for your fast and helpful answer.
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