10. What is Spike-in

I. Spike-in

  • Definition: RNA spike-in is RNA transcripts of known sequence and quantity used to calibrate measurements in RNA hybridization assays, such as microarray experiments, RT-qPCR, and RNA-Seq

  • With the advent of microarray and the commercialization of high-throughput methods for sequencing and RNA detection assays, positive control oligonucleotides are necessary to provide a standard of target sequence concentration. RNA spike-in controls can provide a measure of sensitivity and specificity of RNA-Seq experiment.

  • Origin: mimic eukaryotic RNAs or C. elegans RNA mimic.

  • Usage: A known quantity of RNA spike-in is mixed with the experiment sample during preparation, used as a normalization control for RNA expression profiling. Many researchers choose to spike a synthetic RNA into their RNA prep to monitor RNA recovery and reverse transcription efficiency.

II. Long RNA spike-in kit

  • ERCC RNA Spike-In Mix, ThermoFisher #4456740.

  • 92 transcripts designed to be 250-2000 nt in length.

ERCC RNA Spike-in Mix kit Manuel

ERCC RNA Spike-in Mix kit online

III. Short RNA spike-in kit

Commercial kit

  • ExiSEQ NGS Spike-in kit -- small RNA/microRNA, ExIQEN #800100.

  • 52 synthetic 5' phosphorylated microrNAs of different concentrations.

Quike-guide for ExiSEQ NGS Spike-in kit

Other choices

  • miRNeasy Serum/Plasma Spike-In Control (QIAGEN), a C. elegans miR-39 miRNA mimic.

  • Customized RNA mimics.