I want to inject miRNA from Xenopus sperm with different treatments into fertilized oocytes and watch for any developmental abnormalities. Because I'm new with this kind of techniques I have some questions that maybe some one can answer:
- What is the best protocol or Kit to isolate miRNA?
- Once I isolate the RNA, how can I enrich my miRNA pool? Is there any protocol to amplify sequences without specific primers?, like using universal primers for polymerase reaction.
- What is the best method to deliver the miRNA's? By microinjection, liposomes or another ?
GeneCopoeia offers comprehensive tools for microRNA (miRNA) functional analysis so researchers can detect, express, validate or knockdown microRNA of interest confidently. All known human, mouse and rat microRNA in miRBase covered.
Circulating microRNAs (miRNA) are relatively stable in plasma and are a new class of disease biomarkers. Here we present evidence that high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities. Cellular export of miRNAs to HDL was demonstrated to be regulated by neutral sphingomyelinase. Reconstituted HDL injected into mice retrieved distinct miRNA profiles from normal and atherogenic models. HDL delivery of both exogenous and endogenous miRNAs resulted in the direct targeting of mRNA reporters. Furthermore, HDL-mediated delivery of miRNAs to recipient cells was demonstrated to be scavenger receptor BI-dependent. The human HDL-miRNA profile from normal subjects is significantly different than familial hypercholesterolemia subjects. Notably, HDL-miRNA from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets in cultured hepatocytes. Collectively, these observations suggest that HDL participates in a mechanism of intercellular communication involving the transport and delivery of miRNAs.
I can give you my take on point 1&2:
For the isolation of miRNA you can use any of the commercially available kits from the usual suspects: Ambion, Qiagen, Norgen Biotek etc. Norgen offers many specialized and phenol free kits that are very cost- effective. No matter which kit you decide to go with:
- make sure that it actually retains the miRNA. Sounds obvious but there are a few kits that are called "total RNA" extraction kits but do not retain the miRNAs. Some thing shoudl you decide to use trizol: make sure the protocol is designed to retain the miRNAs.
- once you pick a kit stick with it. While they all work they will all have their particular characteristics resulting in slightly different profiles.
For the amplification you can use Invitrogen's NCode™ miRNA Amplification System kit.
Good luck!
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