Target cRNA Preparation
High quality total RNA is used as starting material to obtain labeled cRNA. In the first step, single stranded cDNA is synthesized by reverse transcription using the poly (A) RNA present in the starting total RNA sample. Single stranded cDNA is then converted into double stranded cDNA and purified using the Affymetrix Cleanup Module. An in vitro transcription (IVT) reaction is then carried out overnight in the presence of biotinylated UTP and CTP to produce biotin-labeled cRNA from the double stranded cDNA. The resulting cRNA is fragmented in the presence of heat and Mg+2 before hybridization to the test array. Due to the high cost of cDNA and cRNA synthesis reactions, we use a very strict quality control measures during the target preparation procedures. At each step the quality of sample is accessed by Agilent Bioanalyzer.
Affymetrix miRNA Galaxy Arrays offer miRNA expression analysis of 71 organisms including Human, mouse and rat. The arrays contain 6,703 miRNA sequences and another 922 sequences that cover human scaRNA and snoRNA. The arrays are used in conjunction the FlashTag Biotin Labeling kit offered by Genisphere (http://www.genisphere.com/array_detection_flashtag_biotin.html). The 45 minute labeling protocol can start with as little as 100ng and also offers an ELOSA QC to ensure proper labeling of the miRNA before it goes onto the array. The samples are washed, stained and scanned using the Affymetrix Command Console and its associated robotics. Metric analyses are carried out using the Affymetrix miRNA QC Tool.
Whole Transcript Arrays
High quality total RNA is subjected to the Ambion Whole Transcript Expression Kit where their patented primer is used to prime for mRNA and coding RNA’s only. The RNAs that have been primed are then amplified to create cDNA and which is used in in vitro transcription to create cRNA. The cRNA is cleaned using Ambion’s bead purification that comes within the WT Expression kit and quantitated. 10ug of amplified cRNA is placed into a reaction with a random primer and then onto to generate a second cycle of 1st strand sense direction cDNA. The cDNA is purified using the Ambion bead cleanup as described previously and quantitated. 5.5ug of sscDNA is then enzymatically fragmented using ADP and UDG using the Affymetrix Terminal Labeling kit and ran on the Agilent bioanalyzer to ensure proper transcript size. The fragmented material is then labeled using TdT and then placed into a hybridization cocktail.