Determination of assay parameters for RNA analysis in bacterial and duodenal samples by spectrophotometry. Influence of sample treatment and preservation.
A simple analytical procedure derived from that described by Zinn and Owens (1980), based on the determination of nucleic purine bases (RNA eq), was carried out to measure microbial nitrogen flow in the ruminant duodenum. Several procedures for sample preservation were tested; the efficiency of each step of the analytical method was also determined using high performance liquid chromatography (HPLC). The proposed method (RNA eq) was compared with two other methods considered as references (2-6 diaminopimelic acid and 35S incorporation) and microbial nitrogen flow was measured in defaunated sheep. The recovery of purine bases analysed by the Zinn and Owens method was generally good (101% pure bases; 90% when bases were added to bacterial samples; 96% when added to yeast RNA). The HPLC measurements allowed us to conclude that this spectrophotometric method is specific for purine bases, all pyrimidine bases being eliminated. Moreover, it was found that the method must be used on freeze-dried samples; storage at + 4 degrees C, defatting or freezing gave incorrect results. Using the described assay, we observed that microbial nitrogen flow at the duodenum of defaunated sheep was not significantly different from that obtained using more traditional markers such as sulphur-35 incorporation or diaminopimelic acid.
K
K
Ushida
B
B
Lassalas
J P
JP
Jouany
D000225:Adenine / Q000032:analysis; D000818:Animals; D001419:Bacteria / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004032:Diet; D004386:Duodenum / Q000032:analysis*; D006147:Guanine / Q000032:analysis; D008297:Male; D012313:RNA / Q000032:analysis*; D012417:Rumen / Q000382:microbiology; D012756:Sheep; D013462:Sulfur Radioisotopes
D013462:Sulfur Radioisotopes; D006147:Guanine; D012313:RNA; D000225:Adenine