瘤胃细菌RNA提取,核糖体RNA去除剂反转录方法.docx

Protocol: Bacterial RNA extraction and transcription1. Rumen fluid sample collection（1） Rumen contents were taken from each cow through rumen fistula and were filtered with four layers of cheesecloth to acquire the rumen liquid.（2） The aliquots of the liquids were dispensed in microtubes and frozen in liquid nitrogen immediately.2. RNA extraction （RNeasy Plus Universal Handbook，Appendix 1）（1） The frozen rumen fluid samples were grinded using Retsch CryoMill。The program are as follows：ProgramTimePrecool10 minCycles5Run time2 minCool time2 minFrequency28/s（2） The grinded rumen fluid samples were then disruption in 1ml QIAzol Lysis Reagent to get a total volume of 1.5 ml。（3） The homogenized samples were vortexed for 2 min to mix the samples and QIAzol well。（4） Place the tube containing the homogenate on the benchtop at room temperature (1525C) for 5 min.This step promotes dissociation of nucleoprotein complexes.（5） Add 100 l gDNA 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. （6） Add 180 l chloroform. Securely cap the tube containing the homogenate, and shake it vigorously for 15 s.Thorough mixing is important for subsequent phase separation.（7） Place the tube containing the homogenate on the benchtop at room temperature for 23 min.（8） Centrifuge at 12,000 x g for 15 min at 4C. After centrifugation, heat the centrifuge to room temperature (1525C) if the same centrifuge will 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, an additional, clear phase may be visible below the red, organic phase. The volume of the aqueous phase should be approximately 600 l.（9） Transfer the upper, aqueous phase (usually 600 l) to a new microcentrifuge tube (not supplied).（10） Add 1 volume (usually 600 l) of 70% ethanol, and mix thoroughly by pipetting up and down. Do not centrifuge. Proceed immediately to step 11.Note: The volume of lysate may be less than 600 l due to loss during homogenization and centrifugation. Precipitates may be visible after addition of ethanol. Resuspend precipitates completely by vigorous shaking, and proceed immediately to step 11.（11） Transfer up to 700 l of the sample to an RNeasy Mini spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at 8000 x g (10,000 rpm) at room temperature (1525C). Discard the flow-through.*Reuse the collection tube in step 12.（12） Repeat step 11 using the remainder of the sample. Discard the flowthrough.*Reuse the collection tube in step 13.（13） Add 700 l Buffer RWT to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 8000 x g (10,000 rpm) to wash the membrane. Discard the flow-through.*Reuse the collection tube in step 14.After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely.*Note: Buffer RWT is supplied as a concentrate. Ensure that ethanol is added to Buffer RWT before use (see “Things to do before starting”, page 17).（14） Add 500 l Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 8000 x g (10,000 rpm) to wash the membrane. Discard the flow-through.Reuse the collection tube in step 15.Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (see “Things to do before starting”, page 17).（15） Add 500 l Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at 8000 x g (10,000 rpm) to wash the membrane.The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions.Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.（16） Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flowthrough. Close the lid gently, and centrifuge at full speed for 1 min.Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin columnafter step 15.（17） Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 3050 l RNase-free water directly to the spin column membrane. Close the lid gently. To elute the RNA, centrifuge for 1 min at 8000 x g (10,000 rpm).（18） Repeat step 17 using another volume of RNase-free water, or using the eluate from step 17 (if high RNA concentration is required). Reuse the collection tube from step 17.If using the eluate from step 17, the RNA yield will be 1530% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.3. Detection of RNA concentrationThe concentration of extracted RNA were detected using Nanodrop 1000, and the ration of OD260/280 were also recorded。4. Electrophoresis of extracted RNA1. The electrophoresis chamber were washed with the DEPC treated water。2. The 1TAE electrophoretic buffers were also prepared using the DEPC treated water。3. Extracted DNA was assessed by agarose gel (1.5%) electrophoresis。4. The DL2000 Marker was used as markers and the electrophoresis was performed at 180 v for about 12min。5. Detection of residual genomic DNA(1) In order to detect if the genomic DNA has been removed completely, a PCR reaction was done as follow。(2) The reaction contains the following： Premix Ex Taq25lRNA1l27f（10mM）2l1492r（10mM）2lAdd dd H2OUp to 50l(3) Condition for PCR reaction:Step 1 (1)94C,5 min Step 2 (30)94C,20 s 55C 40 s 72C 90 s Step 3 (1)72C 10 min 4C For ever (4) PCR products were assessed by agarose gel (1.5%) electrophoresis.6. Remove of rRNA（Ribo-zero-magnetic-kit-(bacteria，Appendix 2)3.A. Preparation of the Magnetic Beads3.A.1. Batch Washing Procedure1. For each Ribo-Zero reaction, 225 l of the Magnetic Beads is required. Note: Mix the Magnetic Beads well by pipetting or gentle vortexing.2. Determine the amount of Magnetic Beads required for the total number of reactions and dispense a maximum of 1,350 l into each 1.5-ml RNase-free microcentrifuge tube (sufficient for six reactions). Pipet the Magnetic Bead suspension slowly to avoid air bubbles and to ensure pipetting of the correct volume. Store unused Magnetic Beads at 4C.Note: When setting up more than six Ribo-Zero reactions, either multiples of 1,350-l aliquots can be washed in a RNase-free 1.5 ml microcentrifuge tubes, or a larger volume can be washed in RNase-free 15-ml tubes (e.g., using a 15-ml magnetic stand).3. Place the 1.5-ml microcentrifuge tube containing the Magnetic Beads on the magnetic stand for at least 1 minute (until the solution appears clear).4. With the 1.5-ml microcentrifuge tube still on the stand, remove and discard the supernatant.5. Remove the 1.5-ml microcentrifuge tube from the stand and add an equal volume of RNase-Free Water. Mix well by repeated pipetting or by vortexing at medium speed.6. Repeat steps 3, 4 and 5 (i.e. wash the beads a total of 2 times with RNase-Free water).7. Remove the 1.5-ml microcentrifuge tube from the magnetic stand. Add a volume of Magnetic Bead Resuspension Solution equal to the number of reactions x 60 l (e.g., for 6 reactions, add 6 x 60 l = 360 l Magnetic Bead Resuspension Solution). Mix well by repeated pipetting or by vortexing at medium speed.Note: The volumes of the beads and Resuspension Solution are additive. Although the washed beads are resuspended in 60 l per reaction, each reaction uses 65 l of resuspended beads.8. Aliquot 65 l of the washed Magnetic Beads into each new 1.5-ml RNase-free microcentrifuge tube (corresponding to the number of Ribo-Zero reactions).9. Optional: Add 1 l of RiboGuard RNase Inhibitor to each tube of resuspended Magnetic Beads, and mix briefly by vortexing.10. Store the microcentrifuge tubes at room temperature until required in Part 3.C.3.B. Treatment of the Total RNA Sample with Ribo-Zero rRNA Removal Solution1. In a 0.2-ml or 0.5-ml RNase-free microcentrifuge tube, combine in the order given:x l RNase-Free Water4 l Ribo-Zero Reaction Buffer1-5 g Total RNA sample (see Table 1)y l Ribo-Zero rRNA Removal Solution (see Table 1)40 l Total volume2. Gently mix the reaction(s) by pipetting and incubate at 68C for 10 minutes. Store the remaining Ribo-Zero rRNA Removal Solutionand Ribo-Zero Reaction Buffer at 70C to 80C. 3. Remove the reaction tube(s) and incubate each at room temperature for 5 minutes.3.C. Magnetic Bead Reaction and rRNA RemovalRequired in Part 3.C: 50C water bath or heating block for 2.0-ml tubes.1. Using a pipette, add the treated RNA from Part 3.B to the 1.5-ml microcentrifuge tube containing the washed Magnetic Beads and,without changing the pipet tip, immediately and thoroughly mix the contents of the tube by pipetting at least 10 times. Then, vortex the tube immediately at medium setting for 10 seconds and place at room temperature. Repeat this process for each sample.Important! Always add the treated RNA sample to the washed Magnetic Beads and immediately mix by pipetting. Never add the Magnetic Beads to the treated RNA sample. Immediate and thorough mixing prevents the beads from forming clumps that can significantly impact the efficiency of the rRNA removal.2. Incubate the 1.5 ml microcentrifuge tube at room temperature for 5 minutes.3. Following incubation, mix the reactions by vortexing at medium speed for 10 seconds and then place at 50C for 5 minutes in anappropriate water bath or heating block. Avoid any significant condensation during this incubation step.4. After the 5-minute incubation at 50C, remove the microcentrifuge tubes and immediately place them on a magnetic stand for atleast 1 minute (until the solution appears clear).5. Carefully remove each supernatant (85-90 l) containing the RNA and transfer to a labeled 1.5-ml RNase-free microcentrifugetube. Important! The supernatant contains rRNA-depleted RNA.Optional: If a small amount of Magnetic Beads are still visible in the supernatant, place the collected supernatant onto the magnetic stand for 1 minute. Remove the supernatant containing the rRNA-depleted RNA and transfer to a new 1.5-ml RNase-free microcentrifuge tube。6. Place the supernatant (RNA solution) on ice and immediately proceed to Part 3.D. Alternatively, the supernatant may be stored at70C to 80C before completing Part 3.D.3.D. Purification of the rRNA-Depleted Sample3.D.3. RNeasy MinElute Cleanup Kit (Cat. No. 74204, Qiagen) Note: RNA purification kits from other suppliers may also be used; however, performance may vary.1. Adjust the sample to a volume of 100 l with RNase-Free Water. Add 350 l of Buffer RLT, and mix well.2. Add 550 l of 96%-100% ethanol to the RNA, and mix well by pipetting. Do not centrifuge. 3. Transfer half of the sample (500 l) to an RNeasy MinElute spin column placed in a 2-ml collection tube (supplied in the Qiagen kit). Close the lid gently, and centrifuge for 15 seconds at 8,000 x g (10,000 rpm). Discard the flow-through. Reuse the collection tube for Step 5.4. Transfer the remaining sample and repeat the centrifugation. Discard the flow-through and collection tube.5. Place the RNeasy MinElute spin column in a new 2-ml collection tube (supplied in the Qiagen kit). Add 500 l Buffer RPE to the spin column. Close the lid gently, and centrifuge for 15 seconds at 8,000 x g (10,000 rpm) to wash the spin-column membrane. Discard the flow-through. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.6. Add 500 l of 80% ethanol to the RNeasy MinElute spin column. Close the lid gently, and centrifuge for 2 minutes at 8,000 x g (10,000 rpm) to wash the spin-column membrane. Discard the flow-through and collection tube.Note: After centrifugation, carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.7. Place the RNeasy MinElute spin column in a new 2-ml collection tube (supplied in the Qiagen kit).8. Open the lid of the spin column, and centrifuge at full speed for 5 minutes. Disc