RNA Preparation and Sample Quality Testing
The Single Cell Genomics Core has the following guidelines for researchers preparing RNA for microarray experiments:
- High-quality RNA is the most important determinant of successful microarray experiments. The utmost care should be taken to provide pure, nondegraded total RNA samples to the core.
- We have found that RNA prepared with Qiagen column systems works well. Care must be taken, however, to remove all residual wash buffer and ethanol from the column before the final sample elution step. We strongly recommend both spinning the column at full speed for at least two minutes, and then applying the column to a Qiagen vacuum manifold system for five minutes to evaporate the remaining ethanol before eluting the sample. Alternatively, one may spin the column for two minutes, and then move the column to a fresh empty tube and spin an additional three minutes.
- If the RNA is isolated using TRIzol, RNAzol, or a comparable procedure, then we strongly recommend using a Qiagen RNeasy cleanup kit to further purify samples.
- For larger tissue samples we recommend immediately freezing with liquid nitrogen, and then grinding to a powder with a mortar and pestle. The powder can then be placed in a TRIzol reagent or other lysis buffer. Placing chunks of tissue into lysis buffer directly often leads to RNA degradation.
- A DNase treatment is strongly recommended. It removes genomic DNA that may interfere with the experiment.
Ideal starting total RNA amounts (not mRNA) depend on the target amplification protocol requested.
- The volume of the sample should not exceed 12 ul.
- The OD 260 / 280 ratio should be at least 1.8 for pure RNA.
- Be sure to clearly label sample tubes. Include a distinct name as well as the concentration of each sample for efficient processing.
Sample Quality Testing
The core staff checks the quality of the submitted RNA or DNA samples using the Agilent 2100 Bioanalyzer. The required concentrations for the QC test are between 100 picograms / microliter - 500 nanograms / microliter. Please submit at least 2 microliters of RNA or DNA per sample in a well labeled 0.5 ml or 1.5 ml tube. If the samples are frozen, I suggest using dry ice for transport. Our facility will store samples in a -80 degree Celsius freezer until the assay is run. If the RNA isolations have not been completed, you may want to create a separate aliquot for the quality control test to minimize the number of freeze-thaws. There is no form that needs to be completed prior to sample submission, however you are required to bring a printout with the name of the samples, concentrations, and your contact information. You are welcome to bring samples to the Single Cell Genomics Core, Monday - Friday between 10:00 A.M. - 5:00 P.M. The lab is in room 3001 in Research building R. The turn-around time is usually 1 -5 business days.
