I am working on some small RNA-Seq data and trying to normalize them. From literature, I found that people are using some housekeeping small RNA as reference for the normalization in qPCR. Can normalization with specific reference RNA sequences also be applied to small RNA-Seq data?
RNA-seq data is different from qPCR. With qPCR you must have an internal, reference gene to define your target gene's expression.
With RNA-seq all the genes are measured at the same time. The only normalisation required is to adjust the library size for comparisons between samples. This is done via the std processing protocols of edgeR or DESeq2 etc.
Hello! It is often better to use the Ambion ERCC controls for normalization, but they have to be spiked-in before you make libraries and sequence. After sequencing you can't do that, but if you use the R/Bioconductor software RUVSeq you can do an empirical normalization which will use genes that are statistically not diferentially expressed. RUVSeq from the bioconductor vignette uses the 5000 least significant DE genes, based on a 20,000 gene mammalian data set, so you would want to scale accordingly. Good luck!
RNA-seq data is different from qPCR. With qPCR you must have an internal, reference gene to define your target gene's expression.
With RNA-seq all the genes are measured at the same time. The only normalisation required is to adjust the library size for comparisons between samples. This is done via the std processing protocols of edgeR or DESeq2 etc.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA