RNA isolation from sperms

RAVAL N.

Junior Member

Join Date: Jan 2015

Posts: 1
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#1

RNA isolation from sperms

01-06-2015, 10:40 PM

Hello,
I am getting very less RNA concentration when extracted from RNEasy Mini kit when measured using Qubit but high yield on Nanodrop, and recently I got almost 8 and 9 260/280 ratio but good 260/230 ratio.....i dont know what this much high ratio indicates.....suggestions would be of great help....Thankx in advance...
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dpryan

Devon Ryan

Join Date: Jul 2011

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#2

01-07-2015, 12:58 AM

In my experience the RNEasy kit doesn't work well at all for sperm RNA extractions. In fact, the only thing we've gotten to work well is Trizol (we're doing mostly small RNAseq on the results and have had good luck with this method). With weird 260/280 ratios like that I wouldn't believe any other value the nanodrop is giving you, they're pretty much guaranteed to be wrong.
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Reply to Nine Things a Sample Prep Scientist Thinks About Before Sequencing

by GATTACAT

Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.
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Yesterday, 11:43 AM
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by SEQadmin2

I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.

Here are nine questions we think about, in roughly the order they matter, before...
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From Collection to Sequencing: Why Sample Preparation and Preservation Define Sequencing Data

by SEQadmin2

Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.

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RNA isolation from sperms

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