Reagents
ReagentsQ. How do I know if the quality of my cRNA is appropriate for the CodeLink™ platform?
A. Quality assessment of the cRNA before fragmentation and hybridization is highly recommended. There are several methods for assessing cRNA concentration, purity and quality and they are described in greater detail in “Protocols for CodeLink™ Control RNA Spikes – Including tips for assessing RNA concentration, purity & quality”. The cRNA should have an A260:A280 ratio of 1.8-2.1. Using denaturing agarose gel analysis, amplified cRNA should appear as a smear from 250 to 5,000 nt. The average size of biotin-labeled cRNA should be approximately 1,400 nt. Using a bioanalyzer, the cRNA profile should display a distribution of sizes from 250-5,500 nt., with most of the cRNA between 1,000-1,500 nt.
Q. What are the storage conditions for CodeLink™ reagents?
A. 5X Fragmentation Buffer: 4ºC
Hybridization Buffer Component A: Room temperature
Hybridization Buffer Component B: 4ºC
Cy5-Streptavidin: Typically shipped at room temperature. Upon receipt, store at 4-6ºC up to the point when you open the bottle. Hydrate contents of the bottle and make aliquots, then store frozen at -20ºC.
CodeLink™ Microarrays: Room temperature in a dry environment.
Q. How much RNA starting material is required to perform a microarray?
A. Applied Microarrays recommends starting with 1 µg of total RNA in the in vitro transcription step of target preparation; however, this will vary by target preparation methodology used and also with the source and quality of the total RNA material.
10ug of prepared cRNA is used per CodeLink™ microarray for hybridization.
Q. Which reagents and equipment are necessary to run CodeLink™ microarrays?
A. Please see the document, Reagents & equipment for CodeLink™ Microarrays.
Q. Do I need to worry about RNase contamination when preparing my RNA samples?
A. Yes. Exercise caution to avoid RNase contamination. All solutions must be RNase-free. Pipette tips must be changed before each step. Use nuclease-free water for all nucleic acid steps. Use aerosol-resistant tips for all pipetting steps.