An improved RNA isolation method based on the acid guanidinium-phenol-chloroform (AGPC) procedure using saline precipitation but no column purification was evaluated for quantifying microbial gene expression using reverse transcription quantitative PCR (RT-qPCR) in rumen contents. The method provided good RNA integrity and quantity extracts. The transcript levels of eight glycoside hydrolase (GH) genes of the major rumen fibrolytic bacterium Fibrobacter succinogenes were quantified in the complex microbiota of a conventional sheep and in a gnotobiotic lamb harboring a microflora containing F. succinogenes S85 as the sole cellulolytic microorganism. This study validated the improved RNA isolation method, RT-qPCR conditions to quantify GH transcripts using either the F. succinogenes S85 tuf gene or the 16S rRNA-encoding gene (rrs) as the reference gene, and demonstrated the need to work with good quality RNAs. Transcripts from all the selected genes cel3, endA(FS), celF and endB endoglucanase genes, cedA cellodextrinase gene, mlg lichenase gene, and xynC and xynD xylanase genes of F. succinogenes S85 were detected and quantified at varying levels in the rumen content of the two animal models. This study opens new perspectives in studying microbial gene expression in the rumen of both conventional and gnotobiotic sheep.