LightCycler 480 qpcr Tools. Meeting the Challenge of Your Research

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Rhoda Bishop
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1 LightCycler 480 qpcr Tools Meeting the Challenge of Your Research

2 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+ ( ) for plasmid DNA: High Pure Plasmid Isolation Kit ( ) Automated processing for cultured cells, whole blood, white blood cells: MagNA Pure LC DNA Isolation Kit I+ ( ) for fresh, frozen, and FFPE tissue: MagNA Pure LC DNA Isolation Kit II (Tissue) + ( ) 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 ( ) see page 4 for HybProbe and SimpleProbe probes: LightCycler 480 Genotyping Master ( ) see page 6 Sequence-independent for generic qualitative and quantitative detection of amplicons: LightCycler 480 SYBR Green I Master ( ) see page 5 for SNP and methylation detection: LightCycler 480 High Resolution Melting Master ( ) see page 6 + For general laboratory use. Products are: For life science research only. Not for use in diagnostic procedures unless indicated otherwise. 2

3 or RNA? RNA Nucleic acid isolation Manual processing for cultured cells and whole blood: High Pure RNA Isolation Kit ( ) for fresh/frozen tissue: + High Pure RNA Tissue Kit ( ) for FFPET: High Pure RNA Paraffin Kit ) Automated processing for cultured cells, whole blood, white blood cells: MagNA Pure LC RNA Isolation Kit High Performance + ( ) for fresh, frozen, and FFPE tissue: MagNA Pure LC RNA Isolation Kit III (Tissue) + ( ) 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 ( ) 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 ( ) see page 6 Enzyme and buffer for efficient transcription of RNA: Transcriptor Reverse Transcriptase ( ) For more information on manual nucleic acid purification visit For more information on automated nucleic acid purification visit For more information on the LightCycler 480 System visit For more information on RealTime ready assays visit 3

4 Rely on Premium Performance of LightCycler 480 Probes and SYBR Green I Masters: LightCycler 480 Probes Master ( ) 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

5 LightCycler 480 SYBR Green I Master ( ) 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 50 ng: * 50 ng: * 50 ng: * 50 ng: * 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,

6 Advantages of further LightCycler 480 Masters: LightCycler 480 High Resolution Melting Master ( ) 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 ( ) For mono- and multiplex genotyping assays Amplicon length: optimal 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 ( ) 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

7 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 More about RealTime ready Custom Assays and Panels at: Additional products Product Catalog Number Pack size LightCycler 480 Instrument II instrument, 96-well version 1 instrument, 384-well version LightCycler 480 Multiwell Plate 96, white plates, with sealing foils LightCycler 480 Multiwell Plate 96, clear plates, with sealing foils LightCycler 480 Multiwell Plate 384, white plates, with sealing foils LightCycler 480 Multiwell Plate 384, clear plates, with sealing foils RealTime ready Cell Lysis Kit 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 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 ( 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

Relative Quantification (Mono-Color) Unknown samples (purified total RNA, mrna or cdna or genomic DNA)

The LightCycler 480 System Short Guide Topic: Purpose: Assay Principle: Detection Format: Result: Relative Quantification (Mono-Color) Describes how to set up and perform mono-color Relative Quantification