Table 2. Estimation of the number of bacterioplankton mRNA molecules in coastal seawater and sequencing depth of the metatranscriptomic libraries.

Calculation method	Sample	mRNA molecules per liter	mRNA molecules per cellb	Sequencing depth (%)
Internal standarda	FN56	1.0 × 1012	238	0.000009
	FN57	0.6 × 1012	142	0.000015
Extracted RNA massc	FN56	0.2 × 1012	48	0.000043
	FN57	0.4 × 1012	95	0.000020
Per cell RNA contentd	FN56	2.6 × 1012	619	0.000003
	FN57	2.6 × 1012	619	0.000003

^aThe libraries contained 4014 (FN56) and 6865 (FN57) copies of the internal standard out of a total of 500 092 (FN56) and 465 519 (FN57) potential protein encoding sequences. The standard was added at 4.7 × 10¹⁰ copies per 5.75 liter of seawater just before cell lysis for total RNA extraction (see Materials and methods for details).

^bCell numbers in the 3 μ filtrate averaged 4.2 × 10⁹ per liter based on epifluorescence microscopy.

^cExtraction yields were 14.4 (FN56) and 32.9 (FN57) μg total RNA from 5.75 liter of seawater. Total RNA is assumed to contain 4% mRNA by mass (Neidhardt and Umbarger, 1996) and bacterial mRNAs are assumed to average 924 nt (Xu et al. (2006)).

^dMarine bacterial cells are assumed to contain 5.7 fg total RNA per cell (mid point of 1.9–9.5 fg range reported by Simon and Azam (1989)). See footnote c for estimate of percent mRNA by mass and footnote b for cell counts per litre.