Removal Of Genomic DNA Contamination During Total RNA Extraction From Salmonella Enterica Samples

August 01, 2016

University of Birmingham
Biosciences
Research Assistant

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Company:

Qiagen

Product Name:

RNase-Free DNase Set (50)

Catalog Number:

79254

During total RNA extraction from the Salmonella enterica (bacterial samples in general), small percentage of genomic DNA is also co-eluted. Presence of genomic DNA in the RNA samples to be used for qPCR or RNA-seq experiments will represent mis quantification of the genes due to presence of genomic DNA. To overcome from this problem, I regularly (in our lab) perform in-column digestion of genomic DNA during total RNA extraction using Qiagen's RNase free DNase enzyme. Such samples were free of genomic DNA which was confirmed with 40 cycles PCR using primers to amplify 1.5 kb gene product. All the samples treated with DNase did not yield PCR product when checked by agarose gel analysis.

Experimental Design and Results Summary

Application

Removal genomic DNA contamination during total RNA extraction

Starting Material

Pelleted RNA samples from S. enterica used in extraction using Qiagen RNeasy midikit method

Protocol Overview

20 microliter of DNase enzyme was mixed with 140 microliter of buffer RDD. Then entire mix was applied to the centre column during RNA extraction steps using RNeasy midikit method as per protocol's instruction. Then allowed to stand at room temperature for 20 minutes. After that, washed with RW1 and RW2 buffer and finally eluted in 200 microliter of nuclease free water.

Tips

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Results Summary

Total RNA samples extracted with the use of DNase treatment were used a template in a PCR reaction to amplify a 1.5 kb gene product along with genomic DNA control. All the samples which were treated with DNase were yielded no PCR product whereas positive genomic DNA control samples yielded 1.5 kb gene product. This diagnostic PCR confirmed the absence of genomic DNA in the total RNA samples extracted using DNase treatment.

Additional Notes

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Summary

The Good

Very simple and easy to use protocol.

The Bad

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The Bottom Line

No RNA extraction kits yield 100 % pure total RNA. It is essential to minimise or remove genomic DNA contamination during total RNA extraction. For this purpose, Qiagen's RNase free DNase enzyme is ideal to use with Qiagen RNeasy midikit method.

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