Eurogentec Headquarters LIEGE SCIENCE PARK • 4102 SERAING • Belgium • Tel: +32 4 372 76 65 Fax: +32 4 372 75 00 • [email protected] • www.eurogentec.com Eurogentec North America 3347 Industrial Ct • Suite A • San Diego, CA 92121 • USA • Tel: +1 858 793 26 61 Fax: +1 858 793 26 66 • [email protected] • www.eurogentec.com Products and procedures described in this protocol are intended for research purposes only. Storage conditions The 18S rRNA Control kit should be stored at -15 °C to -25 °C in a constant temperature freezer. When stored under these conditions the reagents are stable for 1 year. Procedure We strongly recommend preparation of a reaction mix Each 50 |jl reaction requires the addition of 3 jl of 18S Primer Mix (plain cap) and 1 jl of 18S Probe (brown cap). If required a mix containing the appropriate amount of both primer and probe can be prepared. Dispense 4 jl of this mix per 50 jl reaction. The 18S rRNA Control kit contains enough reagents for up to 250 - 50 jl reactions. Reagent Volume Description 18S primer mix 750 jl (plain cap) 18S probe (brown cap) Human Xsomal DNA 500 jl (red cap) 250 jl One tube of 6.25 jM FAM-TAMRA probe One tube of Human male chromosomal DNA at 30 ng/jl in TE* Final concentration of primers is 600 nM. Final concentration of probe is 125 nM. Prepare an appropriate dilution series of chromosomal DNA in TE or PCR grade water and a no template control. The 18S rRNA Control Kit has been developed to detect the human, rat, mouse or rabbit 18S ribosomal RNA gene (cDNA/ gDNA). The obtained amplicon is 121 bp in length, to guarantee an efficient PCR. Program the Real-Time thermocycler using the following recommended parameters: * 30 ng/jl correspond to copies of 10000 genome copy of 18S (1,5 to 2 milion copies of the gene depending on the individual). The 18S rRNA Control kit FAM-TAMRA be combined with Eurogentec Reverse Transcriptase core kit (first strand reaction kit) reference RT-RTCK-05 followed by Eurogentec qPCR MasterMixes, reference RT-QP2X-03 or qPCR Core kits, reference RT-QP73-05. Alternatively a One step RTqPCR MasterMix Plus, reference RT-QPRT-032X can also be combined with the Control kit. Alternatively a qPCR MasterMix (RT-QP2X-03) can be combined with the control kit for quantitation of gDNA. The kit is available with a FAM-TAMRA labelled probe (RT-CKFT-18S) detected in channel for singleplex qPCR and a Yakima Yellow®-Eclipse® DarkQuencher labelled probe (RT- If UNG is used, program the Real-Time thermocycler using the following recommended parameters: UNG activation 2 min. 50 °C HotGoldStar activation / UNG incativation 10 min. 95 °C 40 Cycles 15 sec. 95 °C CKYD-18S) detected in channel 2 for multiplex qPCR.

Other Eurogentec Control Kits available: Eurogentec has developped control kits that can be used for either cDNA or cDNA and gDNA. 18S rRNA Control Kit human, rat, mouse, rabbit cDNA and gDNA very high expression level RT-CKYD-18S or RT-CKFT-18S 23S rRNA Control kit E.coli very high expression level RT-CKFT-23S 28S rRNA Control Kit human, mouse cDNA and gDNA very high expression level RT-CKYD-28S U-actin Control Kit human cDNA and gDNA high expression level RT-CKYD-ACTB GAPDH Control kit human cDNA high expression level RT-CKYD-GAPD U-2-microglobulin Control Kit human cDNA and gDNA high expression...