PCR Technologies Additional Resources

Books, Seminars and Tools

Recommended Textbooks

Recorded Seminars

A series of short seminars covering all aspects of MIQE compliant qPCR has been recorded and available at sigma.com/qpcrseminars.

These seminars include basic qPCR topics and a specific series covering aspects of the MIQE guidelines:

qPCR Topics

MIQE Guidelines

PCR Animation

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Assay Design Tools

Assay design is a worthwhile investment of time and effort. There are several complimentary design services provided by oligo manufacturers and independent websites that support the design of a custom assay and will provide all the information regarding the assay design.

Predesigned Assays

Software

General Information

Sigma-Aldrich provides these links as a courtesy for our customers. Inclusion on this list does not imply endorsement or validation of applications or results.

Sigma-Aldrich will neither collect nor make available any data, analysis or content information.

Products and Support

BioReliance

BioReliance provides testing and manufacturing services to pharmaceutical and biopharmaceutical companies that span the product cycle from early pre-clinical development to licensed production. Our goal is to advance the development and delivery of healthcare and consumer products by providing the highest quality testing, development and manufacturing services in partnership with clients worldwide. For established biotechnology and pharmaceutical companies, outsourcing to BioReliance streamlines product development and reduces time to market. The Company also enables emerging and clinical stage companies who may lack the staff, expertise and financial resources to conduct many aspects of
product development in-house.

For more information, visit
bioreliance.com

CleanAmp™ dNTP

CleanAmp dNTPs are modified nucleoside triphosphates that block DNA polymerase nucleotide incorporation. CleanAmp dNTPs are activated by the initial heating step and subsequent denaturing steps in typical hot start PCR cycling conditions. This process limits the amount of activated dNTPs during each cycle of PCR, allowing for more specific and efficient amplification of the desired product and also reducing or completely avoiding mis-priming or primer dimer formation. CleanAmp dNTPs provide a more affordable solution than hot start enzymes for a variety of PCR-based applications.

Custom DNA and RNA Oligos

As the world’s leading supplier of custom DNA and RNA oligonucleotides, Sigma provides an unrivalled product portfolio, including a wide selection of synthesis scales, modifications and format options to fit all of your PCR and qPCR research needs.

For more information, visit
sigma.com/oligos

Custom qPCR Probes

Whether your application is gene expression analysis, genotyping, pathogen detection, or other, Sigma’s wide selection of reporter and quencher dyes will support all major qPCR detection chemistries, including Dual-Labeled Probes, Molecular Beacons, LightCycler® Probes and Scorpions® Probes. Add Locked Nucleic Acid® (LNA®) to your probe or Molecular Beacon for increased thermal stability and hybridization specificity.

For more information, visit
sigma.com/probes

Extract-N-Amp™ PCR Kits

Sigma’s Extract-N-Amp PCR technology provides all the reagents necessary to extract DNA from a wide variety of cells and tissues and amplify targets of interest by PCR. A novel extraction method eliminates the need for long enzymatic digestions or homogenization. The kit also includes a specially formulated hot start PCR ReadyMix™ for amplification directly from the extract. The kit contains validated protocols to extract and amplify genomic DNA from a variety of starting materials.

For more information, visit
sigma-aldrich.com/extractnamppcrkits

GenElute™ Endotoxin-free Plasmid Prep Kit

This line of kits offers a simple, rapid, cost effective method for isolating endotoxin-free plasmid DNA from recombinant E. coli cultures. Endotoxins (also known as lipopolysaccharides or LPS) are often co-purified with plasmid DNA and significantly reduce transfection efficiencies in endotoxin-sensitive cell lines. High-quality, endotoxin-free DNA is ready for immediate use for the most demanding applications, including transfection into endotoxin-sensitive cells.

For more information, visit
sigma.com/plasmid

GenElute™ Genomic DNA Kits

The GenElute Genomic DNA Purification Kits provide a simple and convenient method to isolate pure, high molecular weight DNA from a variety of mammalian sources. These kits use a silica-based membrane, specially selected for genomic DNA purification. Mini and 96-well formats are available.

For more information, visit
sigma-aldrich.com/genomicdnakits

GenElute™ HP Plasmid Prep Kits

GenElute™ HP Plasmid Prep Kits offer a simple, rapid, cost effective solution for plasmid preparation from E. coli cultures. These kits combine silica-based membrane technology and the convenience of spin or vacuum format. The recovered plasmid DNA is ready for immediate use in applications such as restriction digest, cloning, PCR, transfection and sequencing.
Mini, 96-well, Midi, Maxi and Mega formats are available.

For more information, visit
sigma.com/hpplasmid

KiCqStart® Primers

Compatible with any thermal cycler, KiCqStart Primers areready-to-order, pre-designed primer pairs for quantifying gene expression by SYBR® Green I RT-qPCR. To comply with MIQE, they have been developed using sophisticated bioinformatics tools and validated in silico to avoid off-target amplification and all sequences are provided at purchase. KiCqStart Primers are available as up to three ranked sets of forward and reverse primer pairs for all genes from common model organisms.

For more information, visit
sigma.com/ksprimers

KiCqStart SYBR Green qPCR ReadyMix™

KiCqStart SYBR Green qPCR ReadyMix is a 2× concentrated, ready-to-use reaction cocktail that contains all components, except primers and template, for real-time quantitative PCR (qPCR). This unique combination of proprietary buffer, stabilizers and KiCqStart Taq DNA Polymerase delivers maximum PCR efficiency, sensitivity, specificity and robust fluorescent signal using fast, or conventional, cycling protocols with SYBR Green I qPCR.

Different real-time PCR systems employ different strategies for normalization of fluorescent signals and correction of well-to-well optical variations. It is critical to match the appropriate qPCR reagent to your specific instrument. Sigma offers a variety of reagents to accommodate your instrument requirements.

Cat. No. KCQS00: For Bio-Rad, Cepheid, Eppendorf, Illumina, Corbett, and Roche systems
Cat. No. KCQS01: Low ROX™, for ABI and Stratagene instruments,
Cat. No. KCQS02: with ROX™ for ABI instruments
Cat. No. KCQS03: iQ™, with fluorescein for Bio-Rad systems

LuminoCt® qPCR ReadyMix™

LuminoCt qPCR ReadyMix is designed to deliver unsurpassed assay speeds without sacrificing accuracy, precision, or sensitivity. Inclusion of JumpStart™ Taq antibody in the ReadyMix eliminates polymerase activity at ambient temperatures without causing the performance issues associated with chemically modified hot start Taq. This allows for very rapid activation of the Taq and delivers unparalleled assay sensitivity, while allowing for benchtop reaction setup.

Cat. No. L6669: For fast probe-based quantitative PCR

LuminoCt® SYBR® Green qPCR ReadyMix™

LuminoCt SYBR Green qPCR ReadyMix is designed to deliver unsurpassed assay speeds without sacrificing accuracy, precision, or sensitivity Inclusion of JumpStart™ Taq antibody in the ReadyMix eliminates polymerase activity at ambient temperatures without causing the performance issues associated with chemically modified Hot Start Taq. This allows for very rapid activation of the Taq and delivers assay sensitivity, while allowing for benchtop reaction setup.

Cat. No. L6544: For fast SYBR Green quantitative PCR

MISSION® siRNA

Our MISSION line of siRNA are designed utilizing the Rosetta siRNA Design Algorithm and provide a guarantee of ≥75% knockdown on 2-out-of-3 sequences per gene.

Custom siRNA

High quality and cost-effective custom siRNA sequence design available for your unique or custom sequence through our free in-house design service. Choose from a wide range of modifications, purifications and quantities to tailor to your specific needs.

For more information, visit
sigma.com/customsirna-eu

MISSION esiRNA

MISSION esiRNAs are endoribonuclease-prepared siRNA pools comprised of a heterogeneous mixture of siRNAs that all target the same mRNA sequence. These multiple silencing triggers lead to highly specific and effective gene silencing while providing lower off-target effects. MISSION esiRNA are available individually (pre-designed or custom), as custom arrayed panels, or genome scale libraries that target either human or mouse genes.

For more information, visit
sigma-aldrich.com/esirna

MISSION miRNA Mimics

Human miRNA mimics are available in a ready-to-use miRBase version 17 library or choose from over 1,900 individual mimics. Custom mimics are available for all species, including the current miRBase version content. Our mimics utilize a unique design and modification to significantly reduce possible sense strand off-target effects.

For more information, visit
sigma.com/mimics

MISSION Pre-designed siRNA

The most complete collection of pre-designed siRNA for human, mouse and rat. Multiple sequences are available for every gene, with rankings to indicate performance. Sequences are updated with each version of NCBI RefSeq.

For more information, visit
sigma.com/missionsirna

MystiCq® microRNA cDNA Synthesis Mix

Starting with total RNA, or samples pre-enriched or microRNAs, the MystiCq microRNA cDNA Synthesis Mix kit contains all the components necessary to convert mature microRNAs into cDNA templates for qPCR. Since microRNAs are not naturally polyadenylated, the first step in the cDNA synthesis process is to polyadenylate the microRNAs through a poly(A) polymerase reaction. Next, the ReadyScript® reverse transcriptase, an oligodT adapter primer and other required reagents are added to convert the polyadenylated microRNAs into cDNA. The adapter primer includes a unique sequence at the 5’ end that is complementary to the sequence of the MystiCq Universal PCR Primer. The kit also contains a Positive Control Primer that is specific to SNORD44, a small nucleolar RNA that is widely expressed in most human tissues. Sufficient amounts of the Poly(A) Tailing Buffer and the MicroRNA cDNA Reaction mix are included in the kit to allow for the inclusion of no poly(A) polymerase and no reverse transcriptase control reactions. To analyze individual microRNAs, use the MystiCq Universal PCR Primer (which targets the adapter primer sequence) together with the MystiCq microRNA qPCR Assay Primer and the MystiCq microRNA SYBR Green qPCR ReadyMix.

Cat. No. MIRRT: MystiCq® microRNA cDNA Synthesis Mix

MystiCq® microRNA qPCR Assay Primer

The MystiCq microRNA qPCR Assay Primer is an integral part of the MystiCq microRNA qPCR Assay System. It has been designed to target specific microRNAs with the MystiCq Universal PCR primer and the MystiCq microRNA SYBR Green qPCR ReadyMix on cDNA templates generated by the MystiCq microRNA cDNA Synthesis Mix kit.

For more information, visit
sigma.com/mysticq

MystiCq® microRNA SYBR® Green qPCR ReadyMix™

The MystiCq microRNA SYBR Green qPCR ReadyMix is one of the integral components of the MystiCq microRNA qPCR Assay System. This ReadyMix is a specially-formulated 2× concentrate with an antibody-based Hot Start Taq DNA polymerase and all the optimized components needed for highly specific, reliable and reproducible microRNA qPCR assays. Simply add together the specific MystiCq microRNA qPCR Assay Primer, the MystiCq Universal PCR primer and cDNA template generated by the MystiCq microRNA cDNA Synthesis Kit. Since there are different methods used for fluorescent normalization on different realtime PCR instruments, there are platform MystiCq microRNA SYBR Green qPCR ReadyMixes available.

Cat. No. MIRRM00: Formulated for systems that do not require normalization
Cat. No. MIRRM01: Low ROX™ formulation for miRNA RT-qPCR
Cat. No. MIRRM02: with ROX™, formulation for miRNA RT-qPCR
Cat. No. MiRRM03: Formulation for miRNA RT-qPCR on iQ Bio-Rad platforms

OligoArchitect™ Online

OligoArchitect Online is a free, robust qPCR assay design tool powered by the industry standard Beacon Designer™ platform from PREMIER Biosoft. In addition to providing primer and probe homology searching and template secondary structure analysis, additional features of OligoArchitect Online include automatic retrieval of template sequences, visualization of the template with positional display of regions of interest, design quality ratings and tools for alternative and multiplex designs.

For more information, visit
sigma.com/probedesignonline

OligoEvaluator™

OligoEvaluator is a free, online calculator of oligonucleotide sequence analysis, re-suspension and dilution requirements that is powered by PREMIER Biosoft. In addition to providing information such as melting temperature and molecular weight, additional features include secondary structure analysis and BLAST searching.

For more information, visit
sigma.com/oligocalculator

ReadyScript® cDNA Synthesis Mix

ReadyScript® cDNA Synthesis Mix is a sensitive and easy-touse solution for two-step RT-PCR. This 5× concentrated mix provides all necessary components (except RNA template) for first-strand synthesis including: buffer, dNTPs, MgCl2, primers, RNase inhibitor protein, ReadyScript® reverse transcriptase and stabilizers. ReadyScript® is a RNase H(+) derivative of MMLV reverse transcriptase, optimized for reliable cDNA synthesis over a wide dynamic range of input RNA concentrations. The unique blend of oligo (dT) and random primers in the ReadyScript® cDNA Synthesis Mix works exceptionally well with a wide variety of targets. It is optimized for the production of targets <1 kb in length. ReadyScript® cDNA Synthesis Mix produces excellent results in both real-time and conventional RT-PCR.

Cat. No. RDRT: Complete reagent for first strand cDNA synthesis for RT-qPCR

 

Design Your Probe Online with OligoArchitect™

Sigma is pleased to offer OligoArchitect for all of your primer and probe design requirements. OligoArchitect is complimentary and includes both our online design tool (complete a design by yourself) and our consultative service (have an expert complete a design for you).

Enter the Gateway

Our OligoArchitect Online v4.0 design tool is powered by the industry standard Beacon Designer™ (PREMIER Biosoft).
To start here, visit
sigma.com/probedesignonline

OligoArchitect

When You Need a Consultant

For complex and demanding applications, utilize our consultative service to ensure the success of your PCR, qPCR, or other assays.

With personal consultation from our expert, technical support scientists, your request, including all sequences and data analysis, will be MIQE compliant and usually returned to you within 24 hours.

If required, you will also receive followup assay optimization, data analysis assistance, and troubleshooting support.

When You Need a Consultant

Do Your Own Design

Open the design tool with the click of a button.

Reference documents found here include a Glossary of Parameters and the Exon Design Protocol. Both are available
in PDF format.

This tool has been enhanced for a third time and is now able to create designs with Locked Nucleic Acid® (LNA®).

Do Your Own Design

Login or Register with Us

Are you ready to start? Register as a new user and choose your area.

Returning users can login and proceed.

To start here, visit
oligoarchitect.com

Login or Register with Us

Easy to Use

Type in the name of your assay, enter your sequence, and click Search (see OligoArchitect™ Assay Design).

All reported sequences, associated properties, and assay parameters are available for export to Excel and convenient email ordering.

Easy to Use

Trademarks

The following trademarks and registered trademarks are accurate to the best of our knowledge at the time of publicaton.

Trademarks noted as registered are registered in the United States and possibly other jurisdictions. For the latest information on a specific product, see our website, sigma-aldrich.com, or consult individual manufacturers.

Agilent Technologies, Inc. — Mx3000P®, Mx3005P®, Mx4000™

Applied Biosystems — VIC®

BioMark Technologies Inc. — BioMark™

bioMérieux Société anonyme à conseil d’administration — THxID®

Bio-Rad Laboratories, Inc. — Bio-Rad®, CFX384™, CFX96™, Chromo4™, ddPCR™, Droplet Digital™, iQ™, MiniOpticon™, MyiQ™, Opticon™, QX100™

Bio-Rad QL, Inc — Quantasoft®

Biosearch Technologies, Inc— BHQ®, Black Hole Quencher®, Cal Fluor®, Quasar®

Boehringer Ingelheim International GmbH — GILOTRIF®

Cepheid, Inc. — SmartCycler®

Eppendorf AG — Eppendorf®, Mastercycler®

Excel Scientific, Inc. — ThermalSeal RT2RR™, ThermalSeal RTS™

Exiqon A/S — LNA®, Locked Nucleic Acid™

GE Healthcare — Cy®

GlaxoSmithKline LLC — Mekinist®, Tafinlar®

Lehn & Fink, Inc. — Amphyl®

Life Technologies — Applied Biosystems®, FAM™, HEX™, JOE™, OpenArray®, Oregon Green®, QuantStudio®, QuBit®,
Rhodamine Green™, ROX™, StepOne ™, StepOnePlus™, SYBR®, TAMRA™, TET™, Texas Red®

Microsoft Corporation — Excel®, Microsoft®

Molecular Research Center, Inc. — RNAzol®, TRI Reagent®

OSI Pharmaceuticals, Inc. — TARCEVA®

Polyplus Transfection — ZNA®

Premier Biosoft International — Beacon Designer™

Qiagen Group — Rotor-Gene®, Scorpions®, therascreen®

Quanta BioSciences Inc. — KiCqStart®, MystiCq®

RainDance Technologies, Inc. — RainDrop™

Roche Diagnostics GmbH — MycoTOOL®

Roche Diagnostics Operations, Inc. — cobas®

Roche Molecular Systems, Inc. — LightCycler®, Roche®, TaqMan®

Sigma-Aldrich Co. LLC — BlueView™, DirectLoad™, Extract-N-Amp™, GenElute™, JumpStart™, LuminoCt®, miRPremier®, MISSION®, OligoArchitect™, OligoEvaluator™, Onyx Quencher™, OQ™, ReadyMix™, ReadyScript®, REDTaq®

The Dow Chemical Company or an affiliated company of Dow — Triton®

Thermo Fisher Scientific, Inc. — NanoDrop®

TriLink BioTechnologies, Inc. — CleanAmp™