Isolating RNA from Mammalian Cells for qPCR

April 11, 2017

BlueskyReddit
UC Davis
Biochemistry and Molecular Medicine
Graduate Student Researcher

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Company:

Zymo Research

Product Name:

Direct-zol RNA miniprep kit

Catalog Number:

R2052

Our research goal was to extract total RNA from mammalian cells, and utilize the isolated RNA for quantitative real-time PCR quantification of our target RNA. We chose to use this product because the procedure is quick, only requiring an initial 5 minute incubation of Trizol with cells, along with following the spin-column procedures for an additional 15 minutes. All reagents are provided in the kit, with a very straightforward protocol.

Experimental Design and Results Summary

Application

RNA Isolation

Starting Material

A549 lung carcinoma cells

Protocol Overview

Cell media is aspirated and directly lysed using Trizol. Following incubation, a 1:1 ratio of ethanol is added to the mixture (critical to vortex at this step). The solution is then added to the provided spin column, and washed several times with the provided buffers. Lastly, total RNA is eluted in a 50uL - 100uL volume to yield ~150ng/uL or greater concentrations of total RNA.

Tips

It is more effective to increase incubation time after adding Trizol and frequently mixing the solution by pipetting to completely lyse cells.

Results Summary

The A260/280 and A260/230 ratios were normal to show that the kit was capable of isolating high quality RNA without any ethanol contamination. The RNA was subsequently used for reverse transcription and qPCR. The amount of total RNA isolated was also sufficient to produce three sets of cDNA samples for qPCR.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

The kit is reliable and quick, only takes 15-20 minutes to complete.

The Bad

Trizol is toxic and should be handled with care. Safety glasses and a face mask should be worn during this procedure.

The Bottom Line

We use this product very frequently in the lab as we work with RNA. This kit is also applicable for tissue samples, however, the overall yield of total RNA greatly depends on how fine the tissues are processed (requires lots of grinding).

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