Data from 18 Microsatellite Loci Co-Electrophoresed in a Single Capillary

With Ambion® ERCC Spike-In Controls included in your RNA experiment:

  • Measure sensitivity (lower limit of detection) and dynamic range of an experiment (Figure 1)
  • Quantitate differential gene expression (Figure 2)
  • Ensure quality control amongst experiments

Unlocking the potential of RNA analysis

Data from 18 Microsatellite Loci Co-Electrophoresed in a Single Capillary
Figure 1.

RNA analysis, including gene expression profiling and whole transcriptome surveying, can lead to better understanding of expression patterns in disease states and provides greater insights into biological pathways and molecular mechanisms that regulate cell fate, development, and disease progression. Traditional methods of RNA analysis, such as qRT-PCR and microarrays, are well established but are being replaced by next-generation sequencing, a high-throughput digital alternative. Because each method carries multiple platforms, and with the need to compare various samples across platforms throughout the world, a standard measure for data comparison is necessary. As the capabilities of RNA analysis expand, the necessity to create a standardized view of data will become even more important.

Figure 1. Transcript molar ratios in ERCC Spike-In Mixes The transcripts in Spike-In Mix 1 and Spike-In Mix 2 are present at defined Mix 1:Mix 2 molar concentration ratios, described by 4 subgroups. Each subgroup contains 23 transcripts spanning a 106-fold concentration range, with approximately the same transcript size distribution and GC content.

The controls are ideal for next generation sequencing experiments, such as on SOLiD® System, and supported microarray platforms such as the Illumina® Sentrix® BeadChip.

Setting an RNA standard

Variation in RNA expression data can be attributed to a variety of factors including the quality of the starting material, the level of cellularity and RNA yield, the platform employed, and the person performing the experiment. To control for these sources of variability, a common set of external RNA controls has been developed by the External RNA Controls Consortium (ERCC), an ad-hoc group of academic, private, and public organizations hosted by the National Institute of Standards and Technology (NIST). The controls consist of a set of unlabeled, polyadenylated transcripts designed to be added to an RNA analysis experiment after sample isolation, in order to measure against defined performance criteria. Up until the design of such universally accepted controls, it has been difficult to execute a thorough investigation of fundamental analytical performance metrics.

Figure2.

From the trusted brand of quality RNA reagents, Ambion® ERCC Spike-In Control Mixes are commercially available, pre-formulated blends of 92 transcripts, derived and traceable from NIST-certified DNA plasmids. The transcripts are designed to be 250 to 2,000 nt in length, which mimic natural eukaryotic mRNAs.

Figure 2. SOLiD® System dynamic range and lower limit of detection: Spike-In Mix 1, RPKM values ≥5
A cDNA library was prepared from total RNA containing ERCC Spike-In Mix 1 using the SOLiD® Total RNA-Seq Kit (Cat. No.4445374), then processed and sequenced on the SOLiD® System. The data were normalized to RPKM (reads per kilobase of exon model per million mapped reads; Mortazavi et al. 2008), and filtered using a threshold RPKM value ≥5. Using this filtering criteria, 64 transcripts were detected in the sample containing Spike-In Mix 1.

The filtered RPKM values were plotted as a function of the known ERCC transcript concentrations in Spike-In Mix 1. After adjusting for the volume of Spike-In Mix added to the sample, the sensitivity of this sequencing run, based on approximately 47,000,000 mapped reads, was determined to be ~86 fmol and the dynamic range was determined to be 15.2 log2, or 4.5 log10.

Achieve and compare results with confirmed accuracy

Figure 3.

Ambion® ERCC RNA Spike-In Controls are used to create a standard baseline measurement of RNA both within an experiment and across multiple experiments performed using various samples and platforms. With two spike-in mix formulations (Spike-In Mix 1 and Spike-In Mix 2), various measurements can be examined to assess different parameters in an experiment or across experiments. Measurements are determined via known molar concentrations for each transcript within a spike-in mix and through association of the two mixes (using a combination of ratios across 4 different subgroups of the 92 transcripts. Furthermore, expression fold-change ratios between two samples can be calculated with a high degree of confidence using the highly concordant relationship between ExFold RNA Spike-In 1 and ExFold RNA Spike-In 2 (Figure 3).

Figure 3. SOLiD® System fold-change response: ERCC ExFold RNA Spike-In Mixes, RPKM ≥5
cDNA libraries were prepared from total RNA containing ExFold Spike-In Mix 1 or Mix 2, using the SOLiD® Total RNA-Seq Kit (Cat. No. 4445374), then processed and sequenced on the SOLiD® System. The data were normalized and filtered using a threshold RPKM value ≥5. The observed Mix 1:Mix 2 fold-change ratios were then calculated for ERCC transcripts with ≥ 5 RPKM in both samples. The observed fold-change ratios were transformed into log2 space, then plotted as a function of log2(expected fold-change).

After filtering, 64 ERCC transcripts were included in this analysis, and between 15 and 17 ERCC transcripts per subgroup were detected.

Flexible options for ERCC kit configurations

Ambion® ERCC Spike-In Control Mixes are available in two kit configurations to meet your experimental needs. Use the ERCC Spike-In Mix to determine the dynamic range and lower limit of detection on your platform, and use the ERCC ExFold Spike-In Mixes to assess the accuracy of differential gene expression measurements.

Table 1. ERCC RNA Spike-In Control Mixes: kit configurations.

 ERCC RNA Spike-In Mix 1†ExFold Spike-In Mix 1†ExFold Spike-In Mix 2Nuclease-free Water
ERCC RNA Spike-In Mix
(Cat. No. 4456740)
10 µL--1.75 mL
ERCC ExFold RNA Spike-In Mixes
(Cat. No. 4456739)
-10 µL10 µL1.75 mL

† Although ERCC RNA Spike-In Mix 1 and ExFold Spike-In Mix 1 contain the same formulation of ERCC transcripts, do not substitute ERCC RNA Spike-In Mix 1 for ExFold Spike-In Mix 1 for fold-change assessment. Use only ExFold Spike-In Mix 1 and Mix 2 with the same manufacturing lot number.