Dumbbell-PCR: a method to quantify specific small RNA variants with a single nucleotide resolution at terminal sequences. doi: 10.1093/nar/gkv218.

http://nar.oxfordjournals.org/content/early/2015/03/16/nar.gkv218.full

I've experience with the TaqMan stemloop system (not SYBR green based) as well as SYBR green based methods such as the LNA technology from Exiqon.

Both systems do reliably quantify microRNAs in bodifluids such as Serum. Since miRNA concentrations in Serum are quite low - the following TaqMan protocol allows pre-amplification to increase your sensitivity:

https://tools.thermofisher.com/content/sfs/manuals/cms_094060.pdf

and a paper about a validation study:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244598/

However, since it is always important to validate your identified signals, e.g from an array, I woud prefer the LNA Exiqon System. Recently, there was published a great paper which compared different miRNA profiling methods and the Exiqon system scored quite well:

http://www.nature.com/nmeth/journal/v11/n8/full/nmeth.3014.html

It always depends on which analytical parameter (Sensitivity, reproducibility, etc.) you put your focus.

Concerning RNA isolation I agree with Prabu and would also rcommend the miRNeasy Kit from Qiagen. In my opion it's better than the often used miRVANA Paris Kit.

Good luck with your study.

Johannes

If you are interested in profiling CSF, this paper might also be of interest for you:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126423

what is the best way for RNA isolation from serum? For the price ZYMO DirectZol gives similar results to Qiagen without phase separation.

I also tried DirectZol from ZYMO - however, Ct values from Qiagen were slightly lower in my test results.

In our lab we use the Qiagen miRNeasy serum/plasma kit (Cat.no 217184) for isolation of total RNA including miRNA. We inspect the isolated RNA with the Bioanalyzer, small RNA chip. We have also tried the Zymo DirectZol kit, which worked poorly for our samples.

Dear Marianne

What do you mean by poorly for the Zymo DirectZol.

My Cp_s for 1 microL of a 12 microL rxn of cDNA made with 1.5 microL of RNA isolation.

miR-39      miR-16     miR-155

19.15         19.22        30.76

21.02         20.54        30.81

20.49         20.27        31.34

19.57         20.25        31.15

20.99         19.64        31.17

19.64         21.05        31.64

19.27         23.22        31.24

19.96         22.86        31.21

20.53         22.20        30.67

20.72         22.14        30.74

21.31         22.73        31.16

19.99         20.68        31.24

Dear Fausto,

we isolated miRNA for Next-generation Sequencing, so we did not run qPCR with the Zymo samples. For our purpose, the Qiagen kit worked better.