UVM Microarray Facility at the University of Vermont:
The UVM Microarray Facility staff provides comprehensive support to all projects submitted
including RNA extraction techniques, RNA concentration, and other issues that can present
regarding upfront preparation of samples.
(Microarray Brochure (Mission, Access Guidelines and Prices)
Guidelines for Facility Access and Sample Submission
Step 1: Initial Consultation: All investigators who wish to pursue a project through the facility are strongly encouraged to meet with the Microarray and Bioinformatics Facility staff together to ensure proper experimental design and a clear understanding of sample requirements for processing through the facility including turnaround times. All projects requesting bioinformatic support are required to meet with the bioinformatic staff to draft a design file. Please contact Tim Hunter at 656-2557 or Jeff Bond at 656-4068 to arrange a consultation meeting.
Step 2: Submit Total RNA samples for quality assessment. All RNA samples must be DNase treated prior to submission to ensure all contaminating genomic DNA has been removed. Please submit an RNA quality Assessment Order Form to the intake area of the facility (samples should be placed in the freezer in 305A). It is recommended that you isolate your RNA using TriZol followed by Qiagen's RNeasy kit; although, we have found that in most cases RNeasy works well for cell culture and TriZol for tissue(protocols can be picked up in the facility or downloaded from the website. Information required on the form include RNA concentration (ng/ul), 260/280 ratio, RNA source, and isolation method. Concentration is critical since the dynamic range of the 2100 Bioanalyzer is 10-500 ng/ul for the NanoChip and 200pg/ul-10ng/ul for the PicoChip. We require 2.5 ul of clean RNA for sample assessment. We recommend that you resuspend or elute your RNA in DEP-C water. The facility staff will meet to discuss results and a report including traces will be given back to the investigator and consulted how to proceed. Once you have submitted your samples for Bioanalyzer chip analysis, you are considered to be in the queue for target preparation.
Step 3: Submit RNA samples for Target prep. A Target Prep Form will need to be submitted in the intake area (HSRF 305). Information required is RNA concentration, and A260/280 ratio.
Eukaryotic RNA: The concentration of total RNA [not mRNA] MUST be =20ng/ul. 40-50ng of total RNA is recommended for each sample, but the amount can be modified due to system constraints [see below under limited RNA availability].
Prokaryotic RNA: The required amount of RNA is 10 ug or greater at a concentration NO LESS THAN 500 ng/ul. Alternative methods have allowed the use of 4.5ug but only for special requests. Generally, 4.5-10 ug have generated very good data. For total RNA quantities less than 4 ug, please discuss with the staff. For LCM prokaryotic samples [i.e 100ng], it is possible to synthesize Poly -A tials and amplify with the Nugen Ovation system. This is experimental only and employing this technique will only be performed after serious discussion with the core staff.
**Limited RNA availability: For eukaryotic samples at 200pg to 10ng/ul or total amounts of 1-10 ng, an alternative sample preparation protocol can be used in the microarray facility. The Nugen Pico RNA amplification system has performed very well on LCM samples and other manually micro-dissected tissues. Any RNA at these concentrations is eligible. For prokaryotic samples at less than 250 ng/ul [4ug total], please discuss this with the microarray staff.
FACS Sorting for RNA: Please consult with the microarray staff for successful RNA recovery from cells sorted using flow cytometry. Several key points are highlighted below:
Preparing the flow cytometry is not a small task as it must be completely free of RNases from the sheath tank to the sorting nozzle. This decontamination procedure will take considerable time, so be prepared. Ensure the dip tubes, septa, flow cell, all tubing lines, and nozzles have been completely decontaminated with bleach, RNase ZAP, ethanol, autoclaving, or other qualifying technique prior to the sort. Rinse cytometer with DEPC water. Remember DEPC water DOES NOT inactivate RNases, and is only RNase-free water. The sheath fluid and tank must be RNase free. Use only sterile RNase free tubes on the cytometry that have never been open to contaminated air.
As a control, retain some cells and extract the RNA to determine the condition of RNA prior to the sort. Also before and after trypsinization for adherent cells. Often the flow cytometry, even after a good decontamination procedure can be the source of RNases. Whenever possible suspend cells in RNAlater prior to the sort and after trypsinization. Again, checking RNA integrity at this step is a good control. If RNAlater is not an option, you may consider adding RNase inhibitor to prevent degradation during a sort. Use RNase inhibitors that are DTT independent such as Ambion's Superase-In.
During the sort, sort cell directly into your extraction reagent, something like Trizol LS or Qiagen's RLT. When using TriZol, Only Use TriZol LS which is slightly more concentrated than regular regular TriZol. This formula allows lower quantities of reagent to be used relative to the amount of the sample. (Regular TriZol can tolerate 10 part TriZol to1 part sample volume while TriZol LS can allows 3:1. In the case of Guanidium Isothiocyanate (or Qiagens's RLT buffer) sort at a ratio of 100ul of sort liquid to 350 ul of RLT or a multiple of that.
Immediately after the sort, extract RNA according to the selected reagents manufacturer's protocol and evaluate the integrity of the RNA.
Step 4: Test Chip Hybridization and Scanning. This is optional. The test chip can assess the efficiency of the cDNA reaction and quality of the target prep intensities before moving on to the more expensive target array. An experiment report will be provided to the investigator.
Step 5: Hybridization and scanning of target chip. A DVD will be provided to the investigator including the .dat, .cel, and .chp files. Absolute text files or excel files can be generated upon request. The facility will archive a copy, but we suggest that you also make your own back-up. The facility will generate a report summarizing all steps, quality control checks, and results for potential troubleshooting.
Step 6: Data Analysis: Detailed analysis is the responsibility of the investigator in collaboration with the Bioinformatics Core.
