R T Qpcr Reaction Protocol

General protocol for RT-qPCR reactions to access effect of nicked on T7 transcriptional efficiency #

  1. Prepare nicked templates using NEB provided protocol, available at this link. Use 300 nM sgRNA for digestions. Template should be mix of all plasmids used in downstream analysis. Include Cas9 - sgRNA and untreated controls.
  2. Run agarose gel with 50-100 ng of each sample to demonstrate nicking through shift in nicked band height.
  3. Digest samples protease K phenol / chloroform extact samples.
  4. Transcribe samples with NEB T7 polymerase at ratio of 3 nm template / units T7 Polymerase * 1e6 (see optimizing T7 concentrations for details on this). Terminate transcription by heating to 70C for 10 minutes. Carry out transcription reactions in 20 ul total volume.
  5. Increase reaction volume to 100 ul using appropriate amounts of 10x NEB DnaseI buffer, NEB DnaseI (Rnase free) enzyme and npH20 to digest temlplate DNA in samples. Follow NEB DnaseI protocol.
  6. Digest samples with 2 ul Protease K for 15 minutes at room temperature.
  7. Preform phenol/chloroform extraction of all samples.
  8. Re-suspend samples in appropriate volume of 10 mM Tris-HCl.
  9. Prepare cDNA for qPCR analysis using and following provided protocol for BioRad iScript cDNA Synthesis Kit. Be sure to include a no RT control for each sample to verify DnaseI digestion.
  10. Measure cDNA concentrations. If nessicary dilute samples. It will be best to dilute all samples by sample factor if dilution is needed to make downstream calculations easier.
  11. Prepare qPCR reactions using and following protocol for BioRad iQ™ SYBR Green Supermix using appropriate primers using a set volume of each reaction.
It is critical that the same volume of each transcription reaction be used in qPCR reactions. Since the goal is to measure changes in RNA relative to the un-nicked control at what is effectively one gene the starting RNA masses must not be coordinated in order to give interpretable results.
  1. Calculate fold change between nicked and un-nicked samples for each sgRNA used. Ensure DnaseI digestions were successful.