Quantification Of Total RNA From Salmonella Enterica For Use In RNA-Seq Library Preparation

July 28, 2016

University of Birmingham
Biosciences
Research Assistant

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Life Technologies

Product Name:

Qubit RNA HS Assay kit

Catalog Number:

Q32852

I used Life Technologies fluorescence based quantification kit to quantify total RNA extracted from the S. enterica. This kit is very specific to the type nucleic acids species present in the sample and gives specific and sensitive quantification of RNA. This method is recommended for samples intended to use in sequencing experiments. Final measurement gave me values of 450 ng/microliter total RNA as opposed to the same sample measured in the Nanodrop (520 ng/microliter).

Experimental Design and Results Summary

Application

Quantification of total RNA samples to be used in the sequencing experiments

Starting Material

Total RNA extracted from Salmonella enterica cultures using Qiagen RNeasy miniprep kit

Protocol Overview

First total RNA samples extracted were measured in Nanodrop. Then I diluted original total RNA samples into 1:5 dilution with nuclease free water. This 1:5 diluted samples were used to quantify total RNA using kit's instructions at 1:200 dilution (sample; working buffer). Protocol is very simple and also taking reading (fluorometry) in Qubit Fluorometer is very simple. First run the standard curve using reactions prepared from working buffer with standards provided in the kit. Then run your samples in the Qubit Fluorometer.

Tips

I always recommend diluting original total RNA samples to 1:5 if you get Nanodrop quantification values of 500 ng/microliter or above so that Qubit reading values fall within the dynamic range of standard curve.

Results Summary

Got Consistent Qubit Readings for the samples measured. All of my RNA samples extracted were had 440 to 450 ng/microliter of total RNA.

Additional Notes

None

Related Categories

Image Gallery

Summary

The Good

Very simple and easy to perform protocol. It will take less than 30 minutes to quantify 12 RNA samples at a time.

The Bad

None

The Bottom Line

Quantification of total amount of RNA in the samples which are intended for RNA-seq experiment should be done by Qubit HS RNA assay kit rather than Nanodrop based quantification.

Join the discussion