How much serum would be sufficient for obtaining a good yield?
Hii
For microRNA it depend on the kit of extraction
200 microliter of serum was sfficient to give 4ng as a starter for real time pcr using norgen kit
Hope this help u
You can extend the time of the incubation with isopropyl alcohol. Instead of using 5-10 minutes incubation at room temperature, you can incubate at -20 degrees overnight. This increases the yield of small RNAs.
Trizol has a special reagent called Trizol-LS (liquid samples). It should be better for your purpose although I have not used it personally. 200ul serum was enough for our research. Please note that serum contains inhibitors of PCR and reverse transcription, and using too much RNA template (hence increased amount of co-purified inhibitors) could affect PCR. You may have to pre-amplify target RNA. In our experience, Exiqon's serum RNA extration kit coupled with their RT and LNA primers worked very well using 200ul serum. Good luck !
200 ul of serum should be enough. We have used it for microarray hibridization and qRT-PCR.
For extraction method, I've used miRNeasy kit, I don't have any experience on Trizol alone. Good luck
TRIzol-based technique for serum miRNA??!! strongly not recommended !
I do not recommended trizol for its low yield. But its enough for qPCR. According to my experience, you can use Trizol-ls, and several phenol-chloroform extraction steps to remove the proteins.
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