I want to perform microRNA expression by Real time PCR [SYBR Green Chemistry]. I used Trizol reagent[Ambion] for RNA isolation and RNA quality check in Agarose Gel [ I get intect sharp three band of 28s,18s,5s]. Moreover,i performed 260/280 ratio & it was good RNA quality . so now Is it compulsory to perform RIN number[ Agilent Bioanalyzer] before microRNA expression ? or Any alternative to RIN for RNA integrity check method?
Your RNA should be reasonably intact if you see sharp bands on a gel. Probably you can do PCR without RIN measurement.
I think if you want to be sure, I recommend to run a Agilent system( bioanalyzer 2000
but if you have a control to compare, maybe the results can be considered, probably the editor will ask if you will publish this results.
The gel run you speak of should substitute nicely for a Bioanalyzer 2100 analysis. The 260/280 ratio doesn't tell you anything about quality - only relative purity, so be sure to know that for what it is. The Bioanalyzer looks for the same 'intactness/distinctness' of the 28S, 18S and 5S bands you look for in a gel - so, if you need double confirmation of quality, the Bioanalyzer is the way to go, but, given your good gel result already, the Bioanalyzer may be overkill. The Bioanalyzer (once you have that process down) is easier to run than a gel - so you may want to consider using it in the future. RNA gels are generally a waste of RNA sample. But, if you have a lot of RNA, and RNA Gels are quick and easy for your lab to run, your gel results should be OK, unless you start running into RNA of questionable quality; then the Bioanalyzer can at least give you a tangible "value" for integrity. RINs above 8 are desirable, but RNA with RINs of 6 and above can still be used with fairly good confidence in quantitative applications aimed at relative quantification of smaller amplicons such as the one's you are studying.
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