Hi everyone,

I am having trouble extracting total RNA from gram-negative bacteria (non-typeable Haemophilus influenzae) for RNA-seq. I am following good laboratory practices such as working in a biosafety cabinet, using RNAse away to reduce RNAse contamination, using designated RNA-only equipment (including biosafety cabinet) etc. 

I have been performing RNA extraction using the trizol. The bacteria is grown in 2mL liquid culture using a custom artificial sputum media. Pelleted cells are digested at 37C for 10 minutes using Lysis buffer (20mM Tris-Cl, 2mM EDTA, pH 8.0, 1mg/mL lysozyme, Proteinase K 10mg/mL and 1.2% Triton-X) and then homogenized with 1mL Trizol. 

I have performed the same extraction with 100uL of culture but with Qaigen RNeasy Mini Kit and the RNA concentration is ~100ng/uL. When 2mL is extracted with Trizol protocol my yields are the same or lower than Qaigen kit even though I am using 2mL. (nanodrop and bioanalyzer)

Has anyone had experience with low yields of RNA with Trizol? I have a feeling that my lysis/homogenisation is not adequately disrupting the cells though I'm unsure. I do not see any cell debris in the trizol. 

Thanks,

Amar