REQUIREMENTS FOR SAMPLE SUBMISSION
All samples must be resuspended in RNAse-free water in 1.5 ml RNAse, DNAse free microfuge tubes.
RNA samples for RNAqc on the Bioanalyzer:
Bioanalyzer Quality Control is a process utilizing an Agilent Bioanalyzer 2100. The quality of total RNA, aRNA, cRNA, or mRNA can be checked by placing an aliquot of a sample in the labchip and running the chip in the Bioanalyzer. The Labchip uses capillary electrophoresis to differentiate the RNA on the basis of size.
RNA Quality
High quality total RNA has a very specific trace profile, dominated by the 18S and 28S ribosomal peaks. Any degradation in the sample will be seen as a decrease in size of these peaks and an increase in smaller-sized RNA fragments. Degradation is generally brought about by the action of RNases in the sample, but can also be caused by high temperatures or other denaturing contaminants. A RNA Integrity Number (RIN) is assigned by the bioanalyzer to each total RNA sample. The RIN ranges from 1 to 10, with 10 being perfect quality RNA. The minimum acceptable RIN for labeling and hybridization at the CAT is a 7.
High quality mRNA is generally characterized by a broad swell ranging from several hundred to several thousand base pairs, and can vary considerably depending on what species is being examined and how the mRNA was obtained. There is no equivalent of a RIN number for mRNA.
Sample Volume and Concentration
Two microliters of sample is required for each submission, and submitting 3 ul or more is highly recommended. Miscalibrated pipettors and poorly sealed tubes can contribute to loss of sample, so ensuring that enough sample reaches the CAT is important.
The concentration of the submitted samples must be 25-500 ng/ul. Concentrations less that 25 ng/ul may go undetected by the bioanalyzer, and concentrations over 500 ng/ul may jam the chip.
Bioanalyzer Analysis and Data
The samples will be analyzed within 3 business days of their arrival at the CAT. Once analyzed, a paper and a fax copy will be sent to the submitting researcher. An email will be sent notifying the researcher of the analysis with graphics files of the sample traces attached to the email.
Price
The cost of bioanalyzer analysis is $35.00 per sample for UW-affiliated researchers.
RNA/DNA samples for Target Labeling:
The amount and concentration of RNA needed for target labeling will vary depending on the array platform you choose for your experiments. When submitting a batch of samples, please make sure that all samples are in similar volumes and at similar concentrations to minimize variation in sample treatment during labeling. Samples submitted at lower concentrations than those recommended below may be concentrated in the SpeedVac prior to labeling.
Note that the RNA must be high quality for successful labeling, particularly if you provide a small quantity of sample.
Total RNA samples:
Affymetrix Genechips:
- Total RNA quantity required: > 2 ug if sample is high quality
- Up to 5 ug recommended.
- Concentration: 1 ug/ul if possible
Agilent Arrays:
- Total RNA quantity required: 50 ng if sample is high quality
- Up to 500 ng recommended.
- Concentration: 50 ng/ul if possible
Genomic DNA samples:
Affymetrix 500K SNP Genechips:
- Total DNA quantity required: 250 ng
- Concentration: At least 50 ng/ul
Agilent CGH Arrays:
- Total DNA quantity required: 500 ng
- Concentration: At least 50 ng/ul