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)
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: Submitting Samples for Nucleic Acid Assessment. Please submit an RNA or DNA Quality Assessment order form using your iLabs Account. It is recommended that you isolate your RNA using Trizol followed by Qiagen’s RNeasy Kit. Samples should be DNase-treated. Protocols can be picked up in the facility or downloaded from website. Sample concentration guidelines for Bioanalyzer Nanochips, Picochips, Small RNA, HS DNA, and DNA 1000 chips can be found on the RNA/DNA Quality Assessment forms on our Core iLabs website (https://my.ilabsolutions.com/service_center/show/3129?tab=services). We require 2.5uL of clean sample suspended in nuclease-free water, submitted in 0.5mL tubes with tops labeled.
Step 3: Submitting RNA or gDNA for Target Preparation. Please submit a Target Prep order form using your iLabs Account. The following information is required: Sample ID, RNA/gDNA Concentration (ng/uL), Origination Source, Isolation Technique, and Array Chip to Run. The concentration of RNA samples should be adjusted to 20ng/uL in nuclease-free water, with a 10uL volume submission. ForDNA samples, concentrations should be adjusted to 50ng/uL in T10E0.1, with an 8uL volume submission.
**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. Data will be posted to a secure Microarray Server. A direct link to the data, which includes .CEL, .DAT, and .CHP files, will be emailed to the investigator with instructions for access and to the BioInformatics Core as requested. A DVD copy of the files will be archived at the facility.
Step 6: Data Analysis: Detailed analysis is the responsibility of the investigator in collaboration with the Bioinformatics Core.