I have isolated some blood microRNAs. Can I directly use these miRNAs as a kit to suppress a gene of interest in my cultured cells? Can we use isolated circulating microRNAs to control the expression of a gene of the cultured cells? If so, will their expression be controlled well enough? also one of the most important challenges is the microRNA uptakes, will cells up take these isolated blood miRNAs?
Well miRNAs regulated expression of genes, so to answer your first question look at all the lit. on cancer and miRNA work. They do.... To your second question) look at all the Western Blot data...... To your third ) I dont understand your question. To your fourth) if the cell doesnt express it you will have to inject/transfect depending on your system.
Hi Aref. I really do not understand what you want to do. If you want to downregulate a gene of interes with a miRNA that you have characterice in your cells, why you do not try to clon it in a miRNA-based vector? You could hav a look at the miRNA-Block IT vectors from Invitrogen. I was using them quite extensively in the past and work really good. Perhaps it will take less time and effort to clon your sequence mIRNA into the vector and transfect it than purify the miRNAs from the cells and transfect them.
Dear Chad Haghfill , dear Ruben Deogracias and Dear anna Git ,
thank you very much
I have isolated some blood microRNAs, so may be I have to ask this question in advance : can I directly use these miRNAs as a kit to suppress a gene of interest in my cultured cells?
I don think you can use your isolated miRNAs as a RNAi tool. The main reason is the concentration of your miRNA of interest. Its always best to identify all those circulatory miRNAs and order mimics of those miRNAs for your RNAi study.
Dear Aref Sobhkhizy
If you want to use isolated MicroRNAs as a kit for silencing of genes of interest, it's better to follow three steps:
1. increase concentration of MicroRNA (Isolate them from 10-15cc blood rather than 5cc).
2. select a delivery system with high quality to transfect cultured cells successfully .
3.analyze the expression of your genes of interest with real-time PCR and after that, western blot (to detect levels of protein expression).
I think it is a little hard on the act but I sure you can do it.
good Luck
Of course, you can use some special conditions rather than delivery systems.
Dear Mrs. ravanbakhsh.
thank you for your guidance... may I ask one more question if you don't mind please?
how can I isolate a certain type of microRNA?I mean, there are certainly a large number of microRNAs and I need a certain type in high concentration .
You're welcome.
I think it is better to use MicroRNA capturing procedure by means of probe for a MicroRNA of interest ,somewhat same as cDNA capturing method (if you want just miRNA). For doing it, I think you can use Agilent Haloplex to capture regions of interest Successfully. The protocol is easy to use and cost effective, Of course, I dont know Whether it is possible for you or not. If it is not possible, you can try to change it to cDNA and then amplify by PCR (indirectly procedure). With regard to Low concentration for a certain MicroRNA , It is better amplify it.
Please let me know if you have any questions.
Hi Aref, I have never done this, but in theory this would work. If you know the binding sequence for a certain microRNA, you could produce a fragment through PCR that will bind to the miRNA and attach to a tag. Then you could pour whatever solution contains your miRNA and other things to pull out your miRNA. In order to get the quantity I suspect you want, you will have to do this experiment many times. I dont know if you could turn your miRNA into cDNA and amplify it and turn back into miRNA. I suspect you cant due to the size. Food for thought.
the most important problem is concentration ... dear Chad Highfill , I agree with you, due to size, it's seems to be impossible to turn it back into RNA, I quest it's better to solve the concentration problem by increasing the amount of the blood that is to be isolated ... isn't it? or what's the best performance ? what I have to do?
I would think you would have to greatly increase the blood; thus, you will have to extract what you want from each sample and pool it together
Hi Aref, I've read your question. My concern is : do you want to use specific miRNA to target one specific gene or genes ? Is my understanding correct? if so, I think you should specificaly isolate the specific miRNA that you are interested. but if you do this step, the concerntration of your miRNA will be very low. As Chad said, you had better to amplify it for you further experiment.
What I will suggest is you can try but the feasibility is low, only my opinion.
yes, I want to suppress a gene by using my own extracted microRNAs, but as you mentioned the biggest problem is concentration and I don't know how can I solve the problem?
The first important question is: are your miRNAs mature? premir? primir?
the second question is are your miRNAs in microvesicles or exosomes?
I extensively studied secreted miRNAs and in the exosomes there are often mature miRNAs.
If yours are mature there is no way you can do a targeting experiment.
for the concentration I previously used exoquick.
In my experience the uptake is cell specific, but after the uptake they could pass through the lysosome system.
If you have a known premir with a known target you should do a luciferase targeting experiment.
ap
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