Gene panels were constructed that contained all 5 hairpins targeting a gene along with an empty vector control pLKO.5-nullT and the GFP-targeting hairpin clonetechGfp_437s1c1. cDNA was generated using 10ul of RNA and 10ul of 2x cDNA master mix containing 5x Transcriptor RT Reaction Buffer (Roche), DTT, MMLV-RT (Roche), dNTPs (Agilent; 200415-51), Random 9-mer oligos (IDT), Oligo-dT (IDT) and water. cDNA was diluted 1:9 and quantitative PCR was performed using 250 nM of each primer in 2x Sybr green master mix (Roche) and run on a Roche Light-Cycler 480. Target lincRNA expression and GAPDH levels were computed for each panel. lincRNA expression levels were normalized by GAPDH levels and this normalized value was compared to the reference control hairpins within the panel. Knockdown levels were computed as the average of the fold decrease compared to the two control hairpins. Hairpins showing a knockdown greater than 60% of the endogenous level were considered validated and the best validated hairpin from a lincRNA panel was selected for microarray studies.
