User Guide - ULS Labeling of microRNA
Contents
1. User Guide ULS Labeling of microRNA Version 1 0 February 2009 Product ULS microRNA Labeling Kit Catalog number EA 036 EA 037 and EA 038 Lot number See label on package Unit Size 10 dual color labeling reactions or 20 single color labeling reactions Storage conditions See below IMPORTANT NOTE This user guide contains minimal information to provide experienced users with a short protocol to use at the bench A more detailed full manual can be downloaded from our website http www kreatech com I RNA quality For total RNA isolation we recommend to use Trizol Invitrogen extraction followed by precipitation or microRNA enrichment using any of the following kits miRNeasy procedure QIAGEN mirVana miRNA Isolation procedure Ambion Applied Biosystems miRACLE miRNA isolation kit Stratagene Regardless what source of RNA is being used the following quality criteria should be met z For all RNAs OD2 60 OD229 should be gt 1 90 OD260 ODo29 should be gt 2 10 Be aware that some components in silica based purification systems inhibit the ULS labeling reaction This can be prevented by a final wash step using 80 ethanol PA before elution followed by elution using ultrapure water instead of elution buffer ll ULS Labeling of total RNA or microRNA enriched RNA Note the procedure described below is assuming the need to hybridize 1 ug of total RNA or microRNA enriched RNA per sample on a microarray In2. case your application needs more or less sample to be hybridized you can scale up or down accordingly use a ratio of exactly 1 0 uL of ULS per 1 0 ug of RNA The optimal RNA concentration for ULS labeling is gt 50 ng uL If less than 1 ug RNA is available use lower labeling volumes in order to aim for a RNA concentration of 50 ng uL e g if only 250 ng RNA is available the optimal labeling volume is 5 uL 1 Briefly spin all required reagents to 5 Add 1 0 ul of Cy3 ULS or Cy5 ULS drive the contents off the walls and 6 Incubate sample for 15 minutes at lid 85 2 Pre heat a waterbath OR 7 Place samples directly on ice and thermocycler at 85 spin down to drive the contents off 3 Add RNA and ultrapure water to the the walls and lid tube to a total volume of 17 0 uL 8 Continue with the KREApure 4 Add 2 0 ul of 10 x Labeling Solution purification to remove non reacted to obtain a 1 x Labeling Solution ULS dyes section Ill DIAGNOSTICS Page 1 of 2 K KREATECH Example of Cy ULS labeling of 1 ug of total RNA or microRNA enriched RNA Labeling mixture L 10 x labeling solution Ultrapure water Total volume 20 uL NOTE Be aware that the ratio uL ULS compound vs ug RNA is always exactly 1 1 lll Removal of non reacted ULS dyes using KREApure columns Optional If lower labeling volumes are used adjust volume to 20 uL using ultrapure water 1 Resuspend column material by vortexing 2 Loosen cap turn and snap
3. off the bottom closure 3 Place the column ina 2 mL collection tube 4 Pre spin the column for 1 minute at 20 800 x g i e maximum speed of a typical table top microcentrifuge 5 Discard flow through and column cap but re use collection tube 6 Add 300 uL ultrapure water to the column and centrifuge for 1 min at 20 800 x g 7 Discard collection tube and flow through 8 Place column in a nuclease free 1 5 mL microcentrifuge tube not 9 10 11 12 13 14 Add ULS labeled RNA onto the center of the column bed Centrifuge column for 1 minute at max speed using a tabletop microcentrifuge Flow through contains the purified labeled RNA Determine the degree of labeling DoL by measuring absorbance at 260nm and 550nm for Cy3 ULS or 650nm for Cy5 ULS Calculate the Density of Labeling DoL value using the interactive calculator on our website www kreatech com Store the sample 20 or proceed with the hybridization procedure IV Preparation of labeled total RNA using KREAb ock solution optional provided forhybridization 1 For dual color assays pool the labeled samples 2 Concentrate using a concentrator to nearly dryness This product is intended for RESEARCH USE ONLY IT IS NOT INTENDED FOR DIAGNOSTIC APPLICATIONS and or COMMERCIAL PURPOSES Limited Product Warranty Patent Disclaimer This warranty limits our liability to replacement of this product No other warranti
4. es of any kind express or implied including without limitation implied warranties or fitness for a particular purpose are provided by KREATECH KREATECH shall have no liability for any direct indirect consequential or incidental damages arising out of use or the inability to use this product This product or the use of this product is covered by US patents 5 580 990 5 714 327 5 985 566 6 133 038 6 338 943 and several foreign patents owned by KREATECH Biotechnology B V KREATECH is a trade name of KREATECH Biotechnology B V ULS KREApure and KREAblock are trademarks of KREATECH Biotechnology B V KREATECH s Universal Linkage System are subject to patent protection Cy 3 and Cy 5 are trademarks of Amersham Bioscience part of GE healthcare KREATECH DIAGNOSTICS X 3 KREATECH Diagnostics Vlierweg 20 1032 LG Amsterdam The Netherlands Telephone 31 20 691 91 81 Fax 31 20 696 35 31 e mail info kreatech com website www kreatech com After concentration dissolve the labeled material in 1 4 volume of ultrapure water and add 1 4 volume KREAbDlock optional otherwise add another 1 4 volume of ultrapure water Add 1 2 volume of 2 x Hybridization buffer Distributed by Phalanx Biotech Group 1400 Page Mill Road Building B Palo Alto CA 94804 USA Tel 650 320 8669 Fax 650 320 8488 Email info phalanxbiotech com Website www OneArray com Page 2 of 2
Download Pdf Manuals
Related Search