Table 1.

Comparison of methodological considerations for DNA-SIP, RNA-SIP, PLFA-SIP, and protein-SIP.

Trait	Comparison of applicability of biomarkers	Explanation
Sensitivity	protein > PLFA > RNA > DNA	DNA-SIP requires 15–20% isotopic enrichment, while protein-SIP only requires 1%. RNA labeling is 6.5 faster than that of DNA.
Incubation time	protein > PLFA > RNA > DNA	Incubation time is directly linked to sensitivity. DNA-SIP is the only technique that requires active cell division requiring the longest incubation periods potentially leading to biases.
Taxonomic resolution	DNA ≈ RNA > protein > PLFA	PLFA-SIP only distinguishes broader taxonomic groups, while DNA or RNA-SIP provides identification to the genus level or below. Databases for protein sequences are more limited than for 16S rRNA genes.
Indication of metabolic activity	protein > RNA > DNA	Proteins are the most explicit indicators of metabolic activity, while DNA only shows the metabolic potential.
Ease of isolation	DNA ≈ PLFA > RNA > protein	Isolation of PLFA and DNA are routinely performed in different matrices, but isolation of RNA and proteins from environmental samples can be very challenging.
Stability	DNA ≈ PLFA > protein > RNA	DNA or PLFA are fairly stable, but proteins may denature, and mRNA is very sensitive to degradation.
Application with ’omics‘	DNA > RNA > protein	The application potential depends upon the developmental stage of the ’omics‘ methods. Currently, metagenomics is the most well-developed followed by metatranscriptomics and metaproteomics, respectively.