I am isolating mRNA from leaf tissues for semi-quantitative RT-PCR analysis. I was estimating RNA by spectrophotometrically. But it requires quiet a good amount of RNA. Is there any alternative and good way of estimation process?
If you want to know quantity then use Qubit RNA assays (HS can go as low as 5ng), unfortunately nanodrop gives inaccurate quantification. However, nanodrop can be used to gauge quality, but best is to use Agilent Bioanalyzer RNA chips that'll give you RIN (RNA Integrity Number) quality.
regards
Perhaps looking for , bioanalyser,agilent or experion biorad or caliper perkin elmer wil help in finding a good alternative for the spec. Determination of the quality of your samples
Nanodrop and Qubit RNA assays are good choice but always check it in gel......
I do agree with Edmundo in the perception that Nanodrop overstimates the amount of nucleic acids. In our hands, bioanalyzer understimates the amount of RNA. We have very nice results with quantifications by Qubit when using limited material.
If you want to quantify and check RNA integrity, you should used Bionalayzer.
Dear chandra,,,
First u check the quality of RNA u isolated... may be it is not good enough and less in amount.. check in 1.2- 1.3 % agarose gel... dilute it in DEPC water and use nano drop or Bio photometer for the quantification.. all wl give u the same result.. if ur RNA is not degraded...(before checking worm it in 60-65 degree for 2-5 mint in water bath.... may be this step u skip).. before doing all these thing first u check ur isolation procedure....
all the best...
Greater than 50ng/ul, the nanodrop is good for quantification, less than 50ng/ul a ribogreen assay will be better for quantification. Bioanalyzer chip assays give a good measure of RNA integrity and possible genomic contaminations.
I guess using a nanodrop will do the job for you with as little as 1 - 2 ul of sample..
Nanodrop should be fine even though it can overestimate sometime. you may chose Bioanalyzer if available. to confirm the quality of your mRNA you may look for its integrity and then you should run formaldehyde gel electrophoresis.
Bioanalyzer will give you both the purity , quantity and quality (in terms of integrity).
cheapest method if no access to nano-drop. Densitometry
http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/riboruler-high-range-rna-ladder/
The NanoPhotometer is better than the Nanodrop in many ways. Most importantly, the NanoPhotometer has the best technology to measure small volumes (allowing a minimum of 0.3 uL). No reconditioning or recalibration is necessary with the NanoPhotometer (high quality inert quartz and no path length drift!) which is not the case with the Nanodrop and researchers tend to forget about things like that.
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