RNA Quality Assurance Instructions
Before submitting RNA samples for labeling and hybridization, please follow the procedures below.
RNA must be prepared and examined using the following assays.
- Isolate total RNA using Trizol (GIBCO,BRL) and clean using RNeasy Kit (Qiagen).
When using mRNA it must be isolated from total RNA using Oligotex columns (Qiagen)
or oligo DT cellulose columns (GIBCO,BRL).
- Measure the optical density (OD) of total RNA and mRNA samples including OD260,
OD280, OD260 /OD280, and calculate the concentration (µg/µL)
of your samples. Submit your samples with all the values above and the dilution factor that you
used to take these readings.
For each mRNA sample, please send at least 5 µg at a concentration of 0.5 - 2 µg/µL.
For each total RNA sample, send 12 µg at a concentration 1 µg/µL.
Users outside of UAlbany East Campus must send RNA samples in ethanol on dry ice or as a dry pellet.
- Visualize each total RNA sample by running on an agarose gel containing 1.1% formaldehyde and photograph.
Please attach a photo of the results (please remember to clearly label each lane with the sample label).
Please label each tube of RNA sample prepared in DEPC-H2O carefully and complete all the details on the RNA submission form with the volume and concentration or µg amount for a dry pellet. This form must be faxed to 518-591-7211 or hand delivered to CRC building, Room 328, East Campus, Rensselaer, NY 12144.
Our protocol for RNA Quality Control
To obtain high quality data from your gene chip experiment, it is essential to start with high quality total RNA. If RNA is degraded or otherwise unsuitable for microarray application, investigators will be notified immediately.
A small amount of your sample will be assayed with an Eppendorf BioPhotometer. We require a concentration of at least 1 mg/mL. The OD 260/280 is calculated to estimate the purity of the RNA. A ratio close to 2.00 indicates a high percentage of ribonucleotide.
To assess the integrity of your total RNA, we will test it with the Agilent Technologies 2100 Bioanalyzer Lab-on-a-chip system. This assay is similar to gel electrophoresis in concept, but it is cleaner, more efficient, and only requires a very small amount of sample.
Agilent's Lab-on-a-chip technology
A small amount of sample is loaded into the wells in the chip and electrodes cause the RNA to move through microchannels filled with a sieving polymer and fluorescent dye. Fluorescence signal is plotted against run-time to generate an electropherogram or translated to gel-like images.