FAQs - Microarray Sample Preparation
The quality of array data is directly related to purity and amount of RNA. Our facility has completed extensive research with various purification methods and has compiled a list of suggestions to assist you with producing high quality RNA samples.
There are several RNA purification methods acceptable in producing array quality RNA, but we have found that the most reliable is the use of a glass fiber filter system such as Qiagen's RNeasy mini kit. We have tried other manufacturers’ products, but have gotten varied results. These kits are relatively inexpensive and protocols are easy to follow. A few tips to help with the use of these columns:
- If sample is frozen, be sure not to let it thaw. This will greatly decrease the yield of intact transcripts and could skew your results.
- Homogenization is an extremely important step in RNA isolation. Use a tissue homogenizer or a twenty gauge needle to completely lyse cells.
- Do not overload the column with material. Low purity preps are much less useful than low yield.
- Although the buffers in these kits are optimized to wash DNA off the filter, we have found that many preparation protocols leave enough genomic DNA to skew hybridization, especially among low copy transcripts. We recommend that all samples are DNAse treated, and the DNAse enzyme and buffer removed before experimentation.
- Samples should be concentrated to 20µg in 16.5 µL RNase free water. IF USING SPEED-VAC USE LOW HEAT!! Precipitate RNA using 1/2 volume of 7.5M ammonium acetate and 3 volumes of very cold 100% ethanol (-20deg). RNA does not precipitate well at room temperature, so a 30 minute incubation at -20C is necessary. Centrifuge at maximum g force in a microfuge at room temperature for 20min. Remove the supernatant, and overlay the pellet gently with 250ul ice cold 70% ethanol. Centrifuge at maximum g force for 2 minutes. Remove the supernatant, and dry the pellet for 15-20 minutes at room temperature. Be sure that the pellet is completely dry, but do not over-dry (i.e. speed vac or heat). Resuspend in the appropriate amount of RNAse free water.
- RNA can be stored at -80C for up to one year, but freshly isolated samples are always best. Freeze thawing of RNA can be damaging and should be done as little as possible.
- Another common method used to isolate RNA from difficult tissue is with the use of phenol based reagents like TRIzol or RNAzol. These methods give high yields and purity, but do not remove tRNA and 5s RNA. This can cause inaccuracy in RNA quantitation. Also, residual phenolic compounds can damage reverse transcriptase enzyme, inhibiting the labeling reaction.
To be certain that none of these problems persist, a column purification such as RNeasy is highly recommended following TRIzol preparation.
Quantifying RNA
- RNA can be quantified by U.V. spectrophotometer or through the use of Ribogreen dye (Molecular Probes) and a fluorometer. RNA should also be run on an agarose denaturing gel to verify the integrity of the RNA.
- Purity can be estimated by O.D.260/O.D.280 ratio. A ratio of 1.8 to 2.0 is typical for pure RNA. Buffer pH can affect this ratio, so it is recommended that readings are taken in 10mM Tris, pH 7.2-7.5
- The GATC also offers the Lab on a Chip platform from Agilent. The Agilent Bioanalyzer uses 1ul of RNA to check the integrity and concentration of your sample. This costs $31 for 12 samples and is highly recomended before use in microarray experiments.
- Go back to Microarray FAQs index.
