Measurement of Sensitivity, Dynamic Range and Quantification of Differential Gene Expression

March 08, 2016

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

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Company:

Thermo Fisher Scientific

Product Name:

ERCC ExFold RNA Spike-In Mixes

Catalog Number:

4456739

I have used ERCC ExFold RNA spike in mixes in my bacterial RNA seq library preparation to compare sensitivity, dynamic range and user variation across Illumina Hiseq platform located in two different sequencing centers. Then compared the Spike-In Mix data to known Spike-In Mix concentrations and ratios to assess the dynamic range, lower limit of detection, and fold-change response of your platform. It was found that preparation of libraries and sensitivity was highly consistent within the acceptable variation across two sequencing centers. Dynamic range of detection in both Hiseq platforms in different sequencing centers were in agreement.

Experimental Design and Results Summary

Application

RNA sequencing experiments to measure differential gene expression

Starting Material

Bacterial rRNA depleted total RNA

Protocol Overview

Spike in Mix 1 and 2 were diluted to 1:10 using nuclease free water. 1 microliter of diluted spike in mix 1 or 2 was added to total RNA sample right from the step of ribodepletion protocol. Then ribodepleted RNA samples were used for RNA seq library prep using Ambion's ScriptSeq V2 RNA seq library prep kit. Pooled multiplexed seq libraries were sequenced in two Hiseq platforms in different sequencing centers. Data analysis of differential expression and detection of dynamic range and sensitivity were performed by Spike-In Mix data to known Spike-In Mix concentrations and ratios as mentioned in the kit's instruction manual.

Tips

Randomize spike in mix 1 or 2 addition across the samples

Results Summary

Consistent standard curves of RNA spike in mixes RNA across the two Hiseq platform and all differential gene expression measurements were within the dynamic range of detection in both platforms. We found less variation of RNA seq measurements between two centers.

Additional Notes

None

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Summary

The Good

This kit is very good at detection variation in Differential gene expression measurements across platforms and even across different users performing seq library prep.

The Bad

Highly expensive but needed if large scale RNA seq is to be generated.

The Bottom Line

If the RNA seq experiments are performed at multicenters with different users, then this Kit and use of ERCC ExFold RNA controls is must.

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