Life Technologies
ERCC ExFold RNA Spike-In Mixes
4456739
In our laboratory we routinely use ERCC ExFold RNA spike in mixes while prepping RNA-seq libraries to compare sensitivity, dynamic range and user variation across Illumina HiSeq platforms. We compare 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 sequencing platforms. It was found that preparation of libraries and sensitivity was highly consistent within the acceptable variation across different users and sequencing platforms. Dynamic range of detection in both cases of HiSeq platforms and multiple user prepped samples were consistent and within acceptable range of variation.
High quality differential gene expression measurement through RNA-seq in HiSeq platform
ribosomal RNA depleted Salmonella enterica samples under various stress conditions
This contains two spike in mixes as Spike-In mix 1 and Spike-In Mix 2. 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 were added to total RNA sample right from the step of ribodepletion protocol (randomly across the samples i.e few samples with spike in mix 1 and other few samples with spike in mix 2). Then ribodepleted RNA samples were used for RNA seq library prep using ScriptSeq V2 RNA seq library prep kit. Pooled multiplexed seq libraries were sequenced in two Hiseq platforms. Data analysis of differential expression analysis 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 detailed protocol.
None
Standard curve and Differential gene expression and dynamic range of detection limit and variation across multiple user prepped and between two sequencing platforms were consistent and within the limit of acceptable variation as determined by Spike-in mix data comparison.
Ideal to use this spike in mixes if sequencing center has multiple users and multiple sequencing platforms to account for variability in gene expression data.
Expensive kit
None.