4 replies to this topic

[root]

I'm planning to isolate and clone miRNAs from cells lines under two different conditions - to compare those conditions in regard to which miRNA are specific to a condition.
However, I have no prior experience with miRNA methods.
After doing some readings it is my understanding that there are two main strategies for cloning miRNA: 1. the adaptor ligation
2. using polyadenilation and polyT-adaptor primer for RT reaction.
Which one is better in regards to efficiency?
Which one is easier?
I would greatly appreciate if someone could give me a protocol that works.

Thanks to all
root

[Functional Screens]

If miRNA profiling is what you want to study differential miRNA expression between two cell lines or conditions.  Then, miRNA microarray or realtime RT-PCR might save you time (and troubles as well).

[miRNA man]

Functional Screens, on May 27 2009, 10:16 PM, said:

If miRNA profiling is what you want to study differential miRNA expression between two cell lines or conditions.  Then, miRNA microarray or realtime RT-PCR might save you time (and troubles as well).

Or high throughput sequencing if you want to find novel miRNAs.

[root]

miRNA man, on May 28 2009, 07:43 AM, said:

Functional Screens, on May 27 2009, 10:16 PM, said:

If miRNA profiling is what you want to study differential miRNA expression between two cell lines or conditions.  Then, miRNA microarray or realtime RT-PCR might save you time (and troubles as well).

Or high throughput sequencing if you want to find novel miRNAs.

Thank you for your former answers- so I started looking into this and I'm currently checking costs. So my next question is- for high throughput sequncing- how many biological repeats do I need? and for the microarray?

[Functional Screens]

Genome Res. (2008) 18:610 Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells.

For array, it's up to you to do duplicate, triplicate, or more........