How can I perform miRNA microarrays and real time PCR to validate microarray results? Can I design my own primers for miRNA? Is there any suggested database of miRNA? Can i use SYBR green or Taqman? I'd prefer to use SYBR green as it's cheaper.
http://www.gene-quantification.net/Benes-Castoldi-The-ongoing-evolution-of-qPCR-Methods-2010.pdf
You can use either one, though many people use a Taqman assay.
Qiagen makes a kit for making cDNA specifically from miRNAs, which allows you to either buy primers or design your own (http://www.qiagen.com/products/catalog/assay-technologies/mirna/miscript-sybr-green-pcr-kit).
Our lab uses it with very good results.
I agree with Darrell. Their miRNA assays are specific. The Taqman assay will be much better than SYBR, more sensitive and more specific. Try to cut costs by running a smaller reaction volume. I use an Eco and usually for probe qPCR need 20ul but can sometimes scale down to 10ul.
thx,
I visited life technologies they have kit for reverse transcription which adds sequence that you can buy primer complementary to. and you design the other primer complementary to your target miRNA. you could to those PCR with Sybr green. I used Life Technologies Taqman assays for determining the mRNA level of some cytokines and i must agree with you they`re quite fine and save a lot of time. you do not have to worry about dimer primer, but they are quite expensive as well.
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