LightCycler 480 qpcr Tools. Meeting the Challenge of Your Research

LightCycler 480 qpcr Tools Meeting the Challenge of Your Research

Find the Optimal LightCycler 480 Reagents for Your Research Application: Are you analyzing DNA DNA Nucleic acid isolation Manual processing for cultured cells, whole blood, fresh/frozen tissue, and FFPET: High Pure PCR Template Preparation Kit+ (11 796 828 001) for plasmid DNA: High Pure Plasmid Isolation Kit (11 754 777 001) Automated processing for cultured cells, whole blood, white blood cells: MagNA Pure LC DNA Isolation Kit I+ (03 003 990 001) for fresh, frozen, and FFPE tissue: MagNA Pure LC DNA Isolation Kit II (Tissue) + (03 186 229 001) Which detection format are you using? Real-time PCR Sequence-specific for Hydrolysis Probes like TaqMan, RealTime ready, Universal ProbeLibrary probes, or HybProbe, and SimpleProbe probes: LightCycler 480 Probes Master (04 707 494 001) see page 4 for HybProbe and SimpleProbe probes: LightCycler 480 Genotyping Master (04 707 524 001) see page 6 Sequence-independent for generic qualitative and quantitative detection of amplicons: LightCycler 480 SYBR Green I Master (04 707 516 001) see page 5 for SNP and methylation detection: LightCycler 480 High Resolution Melting Master (04 909 631 001) see page 6 + For general laboratory use. Products are: For life science research only. Not for use in diagnostic procedures unless indicated otherwise. 2

or RNA? RNA Nucleic acid isolation Manual processing for cultured cells and whole blood: High Pure RNA Isolation Kit (11 828 665 001) for fresh/frozen tissue: + High Pure RNA Tissue Kit (12 033 674 001) for FFPET: High Pure RNA Paraffin Kit+ 03 270 289 001) Automated processing for cultured cells, whole blood, white blood cells: MagNA Pure LC RNA Isolation Kit High Performance + (03 542 394 001) for fresh, frozen, and FFPE tissue: MagNA Pure LC RNA Isolation Kit III (Tissue) + (03 330 591 001) Are you performing One- or Two-Step RT-PCR? cdna synthesis Two-Step RT-PCR Ready-to-use kit for efficient transcription for all target RNAs, even those with high GC content: Transcriptor First Strand cdna Synthesis Kit (04 379 012 001) cdna synthesis & Real-time PCR One-Step RT-PCR for Hydrolysis Probes (TaqMan, RealTime ready and Universal ProbeLibrary probes): LightCycler 480 RNA Master Hydrolysis Probes (04 991 885 001) see page 6 Enzyme and buffer for efficient transcription of RNA: Transcriptor Reverse Transcriptase (03 531 317 001) For more information on manual nucleic acid purification visit www.roche-applied-science.com/napure For more information on automated nucleic acid purification visit www.magnapure.com For more information on the LightCycler 480 System visit www.lightcycler480.com For more information on RealTime ready assays visit www.realtimeready.roche.com 3

Rely on Premium Performance of LightCycler 480 Probes and SYBR Green I Masters: LightCycler 480 Probes Master (04 707 494 001) Sequence-specific DNA detection, suitable for qualitative and quantitative analysis and endpoint genotyping Amplicon length: optimal 500 bp, up to 1000 bp High sensitivity and high reproducibility MgCl2 optimization not required Ready-to-use 2 x-concentrated hot start master mix Stable at +15 to +25 C for 24 hours, or at +2 to +8 C for 4 weeks, without loss of efficiency 5' 3'-exonuclease activity dntp Mix prevents carry-over contamination by using dutp instead of dttp Complete assay time approx. 40 min (40 cycles) LightCycler 480 Probes Master performs with excellence for a wide range of assays using the LightCycler 480 Instrument. This robustness reduces the need for optimization, for more convenience and cost effectiveness. Some exemplary results are shown in Figures 1 to 4: Performance comparison LightCycler 480 Probes Master Life Technologies' TaqMan Fast Universal PCR Master Mix LightCycler 480 Probes Master Life Technologies' TaqMan Fast Universal PCR Master Mix 1.93* 1.72* 1.89* 1.86* Figure 1: The LightCycler 480 Probes Master produced better signal dynamics and superior PCR efficiency* in this Beta 2 microglobulin real-time PCR assay. Figure 2: The LightCycler 480 Probes Master produced better reproducibility and signal dynamics in this cofilin 1 real-time PCR assay. LightCycler 480 Probes Master Qiagen's QuantiFast Probe PCR Kit LightCycler 480 Probes Master Takara's Premix Ex Taq 1.89* 1.89* 1.89* no Cq detectable Figure 3: The LightCycler 480 Probes Master produced better reproducibility and signal dynamics in this cofilin 1 real-time PCR assay. Figure 4: The LightCycler 480 Probes Master produced better amplification, PCR efficiency*, reproducibility and signal dynamics in this cofilin 1 real-time PCR assay. 4

LightCycler 480 SYBR Green I Master (04 707 516 001) For qualitative and quantitative studies, as well as for product identification by melting curve analysis Amplicon length: Up to 750 bp, optimal # 500 bp High sensitivity and high reproducibility MgCl2 optimization not required Ready-to-use 2 x-concentrated hot start master mix Stable for 24 hours at +15 to +25 C, or four weeks at +2 to +8 C, without loss of efficiency Total assay time approx. 45 min (40 cycles) 5 min melting curve (optional) LightCycler 480 SYBR Green I Master specifically amplifies and detects a wide range of target assays using the LightCycler 480 Instrument. It is especially suited for amplification of long templates, resulting in high level sensitivity, reproducibility, and specificity. Some exemplary results are shown in Figures 5 & 6 and Table 1: Performance comparison LightCycler 480 SYBR Green I Master Life Technologies' Power SYBR Green PCR Master Mix LightCycler 480 SYBR Green I Master Takara's SYBR Premix Ex Taq II Cq @ 50 ng: 16.9 1.92* Cq @ 50 ng: 21.4 1.73* Cq @ 50 ng: 16.9 1.92* Cq @ 50 ng: 26.2 1.90* Figure 5: The LightCycler 480 SYBR Green I Master produced better PCR efficiency*, specificity, and reproducibility at low concentrations using a 442 bp cytochrome amplicon (CycA). Figure 6: The LightCycler 480 SYBR Green I Master produced better sensitivity, specificity, and reproducibility at low concentrations, using a 442 bp cytochrome amplicon (CycA). Roche Applied Science Life Technologies Qiagen Takara LightCycler 480 SYBR Green I Master Power SYBR Green PCR Master Mix Fast SYBR Green PCR Master Mix QuantiTect SYBR Green Master QuantiFast SYBR Green Master SYBR Premix Ex Taq SYBR Premix Ex Taq II Cq 22.5* Cq 29.6* Cq 25.3* Cq 30.7* Cq 26.5* Cq 37.1* Cq 40.0* Melting Peaks * Cq at highest target concentration (30 ng) Table 1: LightCycler 480 SYBR Green I Master was compared to SYBR Green masters from three other suppliers using a 750 bp -globin amplicon. The LightCycler 480 SYBR Green I Master performed significantly better in sensitivity (see Cq at highest target concentration), reproducibility, and specificity (see Melting peaks). Melting peaks were detected from +65 to +95 C. 5

Advantages of further LightCycler 480 Masters: LightCycler 480 High Resolution Melting Master (04 909 631 001) For the screening of amplified gene fragments for variants, such as SNPs, heterozygosity, methylation Contains ResoLight High Resolution Melting Dye Ideally suited for use in combination with the LightCycler 480 Gene Scanning Software Amplicon length: Up to 500 bp, optimal 300 bp Optimized for highly specific target amplification 2 x-concentrated hot start master mix dntp Mix prevents carry-over contamination by using dutp instead of dttp Total assay time approx. 75 min (45 cycles) LightCycler 480 Genotyping Master (04 707 524 001) For mono- and multiplex genotyping assays Amplicon length: optimal 100-300 bp for monoplex assays, 350 bp for multiplex assays, suitable for up to 750 bp High sensitivity and high reproducibility MgCl2 optimization not required Ready-to-use 5 x-concentrated hot start master mix Stable at +15 to +25 C for 24 hours, or at +2 to +8 C for 4 weeks, without loss of efficiency No 5'-3'-exonuclease activity dntp Mix prevents carry-over contamination by using dutp instead of dttp Total assay time approx. 50 min (40 cycles) LightCycler 480 RNA Master Hydrolysis Probes (04 991 885 001) Includes activator (Mn(OAc) 2) to achieve maximized amplification specificity and efficiency Includes enhancer solution for amplifying difficult (high GC content) templates Amplicon length: Up to 500 bp For analysis of 3 4 target RNAs per assay MgCl2 optimization not required 2.7 x concentrated hot start master mix with Tth DNA Polymerase dntp Mix prevents carry-over contamination by using dutp instead of dttp Total assay time approx. 40 min (45 cycles) 6

Achieve Excellence with Roche's Custom RT-qPCR Assays and Panels: RealTime ready assays are ready-to-use qpcr assays for use in quantification of gene expression in real-time PCR. Each assay includes gene specific primers and a Universal ProbeLibrary (UPL) probe, which is a short FAM-labeled hydrolysis probe containing locked nucleic acid (LNA). Order single assays, choose a focus assay panel, or customize your own panel of assays. All assays are function tested using PCR with speciesspecific cdna on LightCycler 480 Instruments. Stringent criteria for PCR efficiency, sensitivity, and specificity ensure highly reproducible performance under custom conditions. Choose a RealTime ready focus panel that ideally suits your needs, e.g., in cancer research: RealTime ready Human Cell Cycle Regulation Panel RealTime ready Human GPCR Panel RealTime ready Human Apoptosis Panel RealTime ready Human Reference Gene Panel 05 339 359 001 05 353 068 001 05 339 316 001 05 339 545 001 More about RealTime ready Custom Assays and Panels at: http://www.realtimeready.roche.com Additional products Product Catalog Number Pack size LightCycler 480 Instrument II 05 015 278 001 05 015 243 001 1 instrument, 96-well version 1 instrument, 384-well version LightCycler 480 Multiwell Plate 96, white 04 729 692 001 5 10 plates, with sealing foils LightCycler 480 Multiwell Plate 96, clear 05 102 413 001 5 10 plates, with sealing foils LightCycler 480 Multiwell Plate 384, white 04 729 749 001 5 10 plates, with sealing foils LightCycler 480 Multiwell Plate 384, clear 05 102 430 001 5 10 plates, with sealing foils RealTime ready Cell Lysis Kit 06 366 821 001 05 943 523 001 1 kit, 50 lysis reactions with a final reaction volume of 40 µl each 1 kit, 500 lysis reactions with a final reaction volume of 40 µl each Universal ProbeLibrary Set, Human Reference Gene Assays 05 046 114 001 1 set, 100 reactions of 50 µl or 250 reactions of 20 µl for each reference gene HYBPROBE, LC, LIGHTCYCLER, REALTIME READY, RESOLIGHT, SIMPLEPROBE and TAQMAN are trademarks of Roche. NOTICE: Products may be subject to certain use restrictions. Before using a product please refer to the Online Technical Support page (http://technical-support.roche.com) and search under the product number or the product name, whether this product is subject to a license disclaimer containing use restrictions. Products are: For life science research only. Not for use in diagnostic procedures unless indicated otherwise. 7