Hey Everyone,
I would love some input on an issue I'm facing. I'm doing RNA extraction in advance of RT-qPCR / ddPCR analysis.
When I use the QIAGEN RNeasy Plus Mini Kit I'm following the protocol word by word (lysis with RLT + b-ME, gDNA elimination by column, fully drying the RNeasy column after washes etc) and doing all the optional steps. However, I'm getting minuscule yields of RNA from my input which are freshly thawed and rested cryogenically stored neurons.
I've run a range of cell inputs from 0.5 million to 5 million cells and the yields are in the range of 0.4 - 2 µg RNA measured on Nanodrop 2000 directly after elution with RNase-Free water. So a few questions:
Is it possible to be losing material on the gDNA eliminator column?
Should I go back to the RNeasy Mini Kit and do the DNase treatment instead of the gDNA eliminator columns in the plus kit?
Has anyone experienced something like this before? I've done RNA extractions before with this kit on other cell types and it worked alright for RNA-seq applications but this has me perplexed as to why the yield is so low.
Looking forward to your suggestions,
Mike
I've always gotten good yields with the normal RNeasy kit. And if you're really worried about gDNA, just do a post-isolation DNaseI digestion and run the samples through the column a 2nd time. I do this all the time and always have good yields with nearly no gDNA.
I would suggest just going back to the RNeasy Mini Kit if it has worked for you before.
#Source: Trying to extract RNA for qRT-PCR in Leishmania, which has no introns. So I've had to be super careful in my RNA isolations and gDNA elimination.
Hi Yousuf,
Do you inactivate Samples treated with DNAse or directly to to Column second time? If yes, what temperature and how many minutes?
Hello I wanted to give an update on this topic to see if there are further ideas. Ive done subsequent extraction on both live cultured cells (pelleted and aspirated all media and wash in PBS) and snap frozen cells stored at -80oC. The snap frozen cells at the same cell input are giving me much higher RNA yield with very good 260/280 and 230/280 ratios. The live cultured cells give much less (20-50x less) yield. It’s got me very puzzled. I’m wondering if the snap freezing forming ice crystals in the cells is required to rupture the membranes sufficiently. Does anyone have thoughts on this?
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