Got Limited Sample? PreAmp It!
Transcription
Got Limited Sample? PreAmp It!
Got Limited Sample? PreAmp It! PCR Reagents: SsoAdvanced™ PreAmp Supermix and PrimePCR™ PreAmp Assays Bulletin 0000 6576 What is PreAmp? PreAmp provides the ultimate in unbiased, 1,000-fold target-specific preamplification from limited nucleic acids, using up to 100 quantitative PCR (qPCR) or end-point assays and enabling more downstream reactions for screening of respective targets. Why use SsoAdvanced PreAmp Supermix? ■■ Unrivaled, with half the bias specification of the competition ■■ Requires as little as 100 pg of genomic DNA or cDNA ■■ Validated for SYBR® Green and probe chemistries ■■ Optimized for PrimePCR PreAmp Assays ■■ Improved performance using TaqMan and custom assays ■■ Superior sensitivity and efficiency ■■ Economically priced Why use PrimePCR PreAmp Assays? ■■ Available for each PrimePCR gene expression assay ■■ SYBR® Green and probe versions offered ■■ Easy to use — no dilutions needed ■■ Proven performance ■■ ■■ ull wet-lab validation ensures assays meet the minimum F information for publication of quantitative real-time PCR experiments (MIQE) guidelines Optimized for use with SsoAdvanced PreAmp Supermix What types of samples work with PreAmp? ■■ Plant samples ■■ Formalin-fixed, paraffin-embedded samples ■■ Stem cells ■■ Single cells ■■ Small biopsies ■■ Laser capture microdissection samples ■■ Rare samples Visit bio-rad.com/web/PreAmp1 for more information. SsoAdvanced™ PreAmp Supermix and PrimePCR™ PreAmp Assays How do PreAmp products compare to others? A Bio-Rad D 3.00 Twenty-four qPCR probe assays with their respective qPCR reagents were used to evaluate the level of bias. 0.75 –0.75 5 10 15 Number of qPCR assays –3.00 0 25 Bio-Rad Eighty-eight qPCR SYBR® Green assays with their respective qPCR reagents were used to evaluate the level of bias. PrimePCR™ PreAmp SYBR ® Green Assays, SsoAdvanced PreAmp Supermix, and SsoAdvanced™ Universal SYBR® Green Supermix RT 2 Assays, RT 2 qPCR Primer Assay, RT2 PreAMP Pathway Primer Mix, and RT2 qPCR Master Mix Bio-Rad ® Ninety qPCR SYBR Green assays with their respective qPCR reagents were used to evaluate the level of bias. 2.25 1.50 0.75 PrimePCR™ PreAmp SYBR® Green Assays, SsoAdvanced PreAmp Supermix, and SsoAdvanced™ Universal SYBR® Green Supermix 0.00 –0.75 –1.50 PerfeCTa PreAmp 5x SuperMix and PerfeCTa® SYBR® Green FastMix –2.25 22 26 Cq value 10 15 Number of qPCR assays 30 20 25 QIAGEN 3.00 2.25 1.50 0.75 0.00 –0.75 –1.50 –2.25 –3.00 –3.75 –4.50 –5.25 –6.00 –6.75 –7.50 22 24 26 F 3.00 –3.00 18 5 E 3.00 2.25 1.50 0.75 0.00 –0.75 –1.50 –2.25 –3.00 –3.75 –4.50 –5.25 –6.00 –6.75 –7.50 16 18202224 2628303234 Cq value C –0.75 –2.25 BQ score B 20 0.75 0.00 –1.50 TaqMan Gene Expression Assays, TaqMan PreAmp Master Mix, and TaqMan Gene Expression Master Mix –2.25 BQ score 1.50 PrimePCR PreAmp Probe Assays, SsoAdvanced PreAmp Supermix, and SsoAdvanced Universal Probes Supermix 0.00 –1.50 BQ score 2.25 BQ score BQ score 1.50 BQ score 2.25 –3.00 0 Life Technologies 3.00 28 30 Cq value 32 34 Quanta BioSciences 2.25 1.50 0.75 0.00 –0.75 –1.50 –2.25 –3.00 –3.75 –4.50 –5.25 –6.00 –6.75 –7.50 22 24 26 28 Cq value 30 32 34 What is a BQ score? A bias quantification (BQ) score provides a numerical value for the level of bias observed in a PreAmp reaction. It is based on comparing the theoretical difference between a sample with and one without PreAmp compared to the observed (actual) difference between a sample with and without PreAmp. Zero indicates no bias, and the farther from zero the BQ score becomes, the greater the bias introduced. Want more details? Property SsoAdvanced PreAmp Supermix TaqMan PreAmp Master Mix (Life Technologies) PerfeCTa PreAmp 5x SuperMix (Quanta BioSciences) BQ score 90% ≤ 1.50 90% ≤ 0.75 90% ≤ 1.50 Sensitive detection of low-level target genes ••• •• • High efficiency, even for difficult amplicons ••• •• •• PrimePCR PreAmp, TaqMan TaqMan Unknown SYBR® Green and probes Probes SYBR® Green and probes 50 x 50 µl 40 x 50 µl 40 x 50 µl Validated assays Chemistry Number of reactions •Fair ••Good •••Excellent © 2014 Bio-Rad Laboratories, Inc. Bulletin 6576 SsoAdvanced™ PreAmp Supermix and PrimePCR™ PreAmp Assays How does it work? From RNA to qPCR results, data can be obtained in less than 5 hours using our validated reverse transcription quantitative PCR (RT-qPCR) products. 1 Extract RNA 2 3 Produce cDNA Combine components 4 Perform preamplification PCR cDNA Reverse transcription with iScript™ Reverse Transcription Kits 5 Dilute sample 1:5 to 1:20 6 Preamplified sample Set up qPCR reaction Primers 7 10 –12 cycles SsoAdvanced PreAmp Supermix Perform qPCR SsoAdvanced Universal Supermix 8 Analyze data 104 RFU Pooled primers 103 102 0 1020 3040 Cycles Preamplification workflow. How do I order SsoAdvanced PreAmp Supermix? Order online at bio-rad.com/web/PreAmpSmx, or contact your local sales representative to request a quote for SsoAdvanced PreAmp Supermix (catalog #172-5160). How do I order PrimePCR PreAmp Assays? Order online at bio-rad.com/web/PrimePCRPreAmp, or contact your local sales representative for additional information. What other validated reagents do I need? iScript cDNA Synthesis Kits provide unbiased representation of target mRNA levels through the use of an RNase H+ Moloney murine leukemia virus (MMLV) reverse transcriptase and optimal priming methodology using a blend of oligo(dT) and random primers. iScript kits are available in single- or multiple-tube formats ■■ — Order online at bio-rad.com/web/iScriptKits, or contact your local sales representative for additional information ■■ soAdvanced Universal Supermixes employ our patented* Sso7d fusion enzyme technology for higher processivity, S increased PCR inhibitor tolerance, and robust performance with challenging templates and target sequences for all real-time PCR applications and all real-time PCR systems. SsoAdvanced Universal Supermixes are available in SYBR® Green and probe versions — Order online at bio-rad.com/web/SsoAdvanced, or contact your local sales representative for additional information * U.S. patents 6,627,424; 7,541,170; and 7,560,260. © 2014 Bio-Rad Laboratories, Inc. Bulletin 6576 SsoAdvanced™ PreAmp Supermix and PrimePCR™ PreAmp Assays PerfeCTa is a trademark of Quanta BioSciences, Inc. RT2 is a trademark of SABiosciences Corporation. SYBR is a trademark of Life Technologies Corporation. Bio-Rad Laboratories, Inc. is licensed by Life Technologies Corporation to sell reagents containing SYBR Green I for use in real-time PCR, for research purposes only. TaqMan is a trademark of Roche Molecular Systems, Inc. Bio-Rad’s real-time thermal cyclers are covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 6,767,512 and 7,074,367. The use of SsoAdvanced Supermixes and PrimePCR PreAmp Assays is covered by one or more of the following U.S. patents and corresponding patent claims outside the U.S.: 5,804,375; 5,538,848; 5,723,591; 5,876,930; 5,994,056; 6,030,787; 6,171,785; and 6,258,569. The purchase of these products includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, are conveyed expressly, by implication, or by estoppel. These products are for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Bio-Rad Laboratories, Inc. 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