瘤胃细菌RNA提取,核糖体RNA去除剂反转录方法.docxVIP

Protocol:Bacterial RNA extraction and transcriptionRumen fluid sample collectionRumen contents were taken from each cow through rumen fistula and were filtered with four layers of cheesecloth to acquire the rumen liquid.The aliquots of the liquids were dispensed in microtubes and frozen in liquid nitrogen immediately.RNAextraction（RNeasy? Plus Universal Handbook，Appendix1）The frozen rumen fluid samples were grinded using RetschCryoMill。The program are as follows：ProgramTimePrecool10 minCycles5Run time2 minCool time2 minFrequency28/s The grinded rumen fluid sampleswere then disruption in 1ml QIAzol LysisReagent to get a total volume of 1.5 ml。The homogenized samples were vortexed for 2 min to mix the samples and QIAzol well。Place the tube containing the homogenate on the benchtop at roomtemperature (15–25°C) for 5 min.This step promotes dissociation of nucleoprotein complexes.Add 100 μlgDNA Eliminator Solution. Securely cap the tube containing the homogenate, and shake it vigorously for 15 s.Addition of gDNA Eliminator Solution will effectively reduce genomic DNA contamination of the aqueous phase, making further treatment with DNase unnecessary.Add 180 μl chloroform. Securely cap the tube containing the homogenate, and shake it vigorously for 15 s.Thorough mixing is important for subsequent phase separation.Place the tube containing the homogenate on the benchtop at roomtemperature for 2–3 min.Centrifuge at 12,000 x g for 15 min at 4°C. After centrifugation, heatthe centrifuge to room temperature (15–25°C) if the same centrifugewill be used in the later steps of this procedure.After centrifugation, the sample separates into 3 phases: an upper,colorless, aqueous phase containing RNA; a white interphase; and a lower,red, organic phase. For tissues with an especially high fat content, anadditional, clear phase may be visible below the red, organic phase. Thevolume of the aqueous phase should be approximately 600 μl.Transfer the upper, aqueous phase (usually 600 μl) to