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Hi!
We're currently trying to synthesize cDNA from total RNA extracted from tissue. Our protocol starts with the Trizol reagent, the Oligotex mRNA mini kit, the NEB protocol for mRNA fragmentation, and then a mixture of a bunch of different protocols for the cDNA synthesis/end repair/dA-tailing/adaptor ligation.
We used a Nanodrop (not the best, we know, but it's all we have) to see how much RNA we had, and it wasn't very much. We went on with the protocol anyway, and ended up with extraordinarily little cDNA (which shouldn't really be a surprise, given the mRNA starting amount).
We were hoping for advice on how to increase the amount of either mRNA or cDNA that we get. We're considering:
-NuGen Ovation (may be a bit too expensive..)
-DSN Normalisation
-Whole transcriptome amplification
Any other suggestions or feedback on those three would be extremely helpful!
We're currently trying to synthesize cDNA from total RNA extracted from tissue. Our protocol starts with the Trizol reagent, the Oligotex mRNA mini kit, the NEB protocol for mRNA fragmentation, and then a mixture of a bunch of different protocols for the cDNA synthesis/end repair/dA-tailing/adaptor ligation.
We used a Nanodrop (not the best, we know, but it's all we have) to see how much RNA we had, and it wasn't very much. We went on with the protocol anyway, and ended up with extraordinarily little cDNA (which shouldn't really be a surprise, given the mRNA starting amount).
We were hoping for advice on how to increase the amount of either mRNA or cDNA that we get. We're considering:
-NuGen Ovation (may be a bit too expensive..)
-DSN Normalisation
-Whole transcriptome amplification
Any other suggestions or feedback on those three would be extremely helpful!