Vendor The Scientist Brand Sigma-Aldrich Version MRK And .

RT-PCR / RT-qPCRReverse transcriptase PCR (RT-PCR) is a variation of PCR which employs reverse transcriptase inaddition to PCR reagents to amplify a specific mRNA sequence. Following the production of a cDNAcopy, which anneals to one of the primers leading to first strand synthesis, PCR/qPCR ensues togenerate dsDNA.One-StepTitan One-Tube RT-PCR SystemDuring one-step RT-PCR / RT-qPCR, the reversetranscriptase step is included in the same tube asthe PCR reaction, facilitating multiplex analysis ofthe gene of interest and the control within a singlewell. One-step reactions are easy to set up, requiringminimal hands-on time to reduce possible errors andcontamination, and are often considered the bestoption for high throughput screening. They are alsoideally suited to workflows which require repeatedperformance of only a small number of assays.Transcriptor One-Step RT-PCR KitContaining four different enzymes, the TranscriptorOne-Step RT-PCR Kit is designed for fast, sensitive, andspecific end-point RT-PCR analysis of RNA. TranscriptorReverse Transcriptase ensures sensitive and robustreverse transcription with high yield. Protector RNaseInhibitor provides maximum template protectionduring reverse transcription, and the Expand System– a blend consisting of Taq DNA Polymerase and aproofreading polymerase – minimizes the possibilityof mutations to afford high yield and fidelity within thePCR.The Titan One Tube RT-PCR System uses ReverseTranscriptase AMV for first strand cDNA synthesis andthe Expand High Fidelity enzyme blend consistingof Taq DNA Polymerase and a polymerase with aproofreading activity for amplification of cDNA by PCR.Facilitating the cloning of rare messages without theneed to construct cDNA libraries, the Titan One TubeRT-PCR System reduces the error rate in PCR throughthe proofreading capability. Sensitive, rapid and reproducible analysis of RNA Threefold higher fidelity in comparison to Taq DNApolymerase Detect as little as 1 fg of total RNA Exceptional sensitivity due to high enzyme efficiency Includes a superior RNase inhibitor, fully active atelevated temperaturesTwo-Step Proprietary hot start buffer for high specificity andreduction of primer-dimersDuring two-step PCR, cDNA is created by reversetranscription and is then added to the PCRreaction. This highly sensitive technique is typicallyrecommended for reactions in which maximumperformance of reverse transcription and PCR isrequired, and relies on separate, optimized buffers foreach step. Affording the possibility to store cDNA forlater use, two-step PCR is often chosen for the study ofmultiple targets per sample.Figure 1: Transcribe difficult templates with high sensitivity.(A) Various amounts (down to 1 fg) of HeLa total RNA were reversetranscribed with the Transcriptor One-Step RT-PCR Kit. A 389 bpfragment (GC content is 64%) was amplified with specific primers forhuman 28S ribosomal RNA according to the kit’s standard RT-PCRprotocol (reverse transcription at 50 C for 30 minutes). As shown bythe clearly visible band obtained after agarose gel electrophoresis andethidium bromide staining, the kit transcribes even small amounts (1fg) of template with high sensitivity.Figure 2: Detection of mouse β-actin mRNA by two-step RT-PCR on aconventional thermal cycler.Transcriptor Reverse TranscriptaseRecommended for long targets (up to 14 kb), raretargets and GC-rich sequences, Transcriptor ReverseTranscriptase is also suitable for the preparation of7

labeled cDNA since it accepts a wide variety of modifiednucleotides. Delivering high sensitivity in two-stepRT-PCR, Transcriptor Reverse Transcriptase may beused in conventional thermal cyclers and real-time PCRinstruments.First Strand cDNA Synthesis Kit for RT-PCR (AMV) Generate cDNA libraries with large inserts Operational at high temperatures (up to 65 C) Suitable for Cy3, Cy5, DIG, biotin, and aminoallyllabeling during cDNA synthesisReverse Transcriptase AMVA gene product of the RNA genome of avianmyeloblastosis virus, the mature form of ReverseTranscriptase AMV includes an RNA-directed DNApolymerase, a DNA-dependent DNA polymerase, anRNase H, and an unwinding activity. The enzyme canbe used for cDNA synthesis, synthesis of first strandcDNA for use in subsequent amplification reactions,dideoxy DNA sequencing, RNA sequencing, 3′ endlabeling of DNA fragments, and the generation of ssprobes for genomic footprints. Suitable for multiple applications For transcripts with difficult secondary structure Functionally tested for cDNA synthesis and RT-PCRTranscriptor Universal cDNA MasterContaining a unique enzyme with a broad temperaturerange making it suitable for high temperature reversetranscription, the Transcriptor Universal cDNA Masterproduces high specificity and sensitivity, even withcomplex RNA templates. With all the necessaryreagents for two-step PCR supplied in just two vials tominimize pipetting, the Transcriptor Universal cDNAMaster can be combined with the RealTime ready CellLysis Kit for direct use with cell lysates. Effortless setup simply requires the addition of RNA Compatible with incubation temperatures up to 65 C Suitable for target amounts as low as 0.1 pg totalRNA8The Complete Roche PortfolioSuitable for use with sequence-specific primers,poly(dT)15 primers or random primers, the First StrandcDNA Synthesis Kit is used to generate first strandcDNA as the starting reaction for two-step RT PCR. Thehigh thermostability of the avian myeloblastis virus(AMV) reverse transcriptase enzyme allows the reactionto be performed at 42 C, giving higher specificity andsuperior resolution of secondary structures. Thermostable enzyme affords enhanced resolution ofsecondary structures Compatible with a sequence-specific primers,poly(dT)15 primers, or random primers Can be used in combination with PCR for detection ofpresence or absence of RNA virusesCat. No.DescriptionTOSRTROTranscriptor One-Step RT-PCR Kit11855476001Titan One-Tube RT-PCR SystemTRANSRTROTranscriptor Reverse TranscriptaseTOSRTROTranscriptor One-Step RT-PCR Kit11855476001Titan One-Tube RT-PCR SystemTRANSRTROTranscriptor Reverse Transcriptase10109118001Reverse Transcriptase AMV05893151001Transcriptor Universal cDNA Master11483188001First Strand cDNA Synthesis Kit for RT-PCR(AMV)

DIG-LabelingProviding an environmentally friendly and safer alternativecompared to radioactive materials, the DIG System is the nonradioactive technology of choice to label and detect nucleic acids.Based on the steroid digoxigenin (DIG), DIG-labeled probes affordhigh sensitivity and low background across multiple applications.Furthermore, since DIG antibodies do not bind other substrates,DIG offers exceptional specificity.There are two main objectives of the DIG system. Both methodsrequire labeling of the desired DNA and RNA probes first. M embrane hybridization: using membrane or blots to detectDNA or RNA. In Situ hybridization: performing in situ localizationDIG DNA Labeling by PCRFor both membrane and in situ hybridization, PCR is auseful labeling method. DIG-labeled probes are idealfor PCR applications in which the template is availablein only limited amounts, is partially purified, or is veryshort. The PCR DIG Probe Synthesis Kit contains analkali-labile DIG-11-dUTP formation, enabling simpleremoval of the DIG label following chemiluminescentdetection and subsequent re-hybridization of blots withmultiple DIG-labeled probes.Detection of a single-copy gene (Β-actin) intotal human DNA using the standard protocol.DIG Random Primed DNA LabelingDIG Oligonucleotide LabelingDuring random primed DNA labeling, the DNA templateis copied in the presence of hexameric primersand alkali-labile DIG-11-dUTP. Suitable for labelingtemplates of almost any length, this technique isespecially suitable for single copy gene detection ongenomic Southern blots and in screens of recombinantlibraries.DIG-labeled synthetic oligonucleotides are excellenthybridization probes for in situ hybridization, dot/slotblots, library screening, and the detection of repeatedgene sequences on Southern blots. Several methodsare available for DIG-labeling of oligonucleotides,including 5 end-labeling with DIG-NHS-Ester, 3 endlabeling with DIG-ddUTP and the addition of a 3 tailconsisting of DIG-dUTP and dATP.Nick Translational LabelingNick translation is commonly used to label probes forin situ hybridization. Based on the ability of DNase I tointroduce randomly distributed nicks into DNA, whichare subsequently used as primers for DNA synthesis byDNA polymerase I, nick translation can be employed toincorporate DIG-11-dUTP into newly synthesized DNA.The method of choice for in situ hybridization, nicktranslation produces highly sensitive targets for indirect(immunological) detection.Transcriptional Labeling of RNA ProbesCat. No.Description11636090910PCR DIG Probe Synthesis Kit11277073910DIG RNA Labeling Mix11175025910DIG RNA Labeling Kit (SP6/T7)11745832910DIG-High Prime DNA Labeling and DetectionStarter Kit I11585614910DIG-High Prime DNA Labeling and DetectionStarter Kit II11603558001DIG Easy Hyb 11585762001DIG Wash and Block Buffer SetFor some applications, DIG-labeled RNA is a moreeffective hybridization probe than DIG-labeled DNA.One example is its utility to detect rare mRNAs withinnanogram amounts of total RNA. In vitro transcriptionfrom a DNA template facilitates the production of largequantities of full-length DIG-labeled RNA copies.9

ProteomicsProtein StabilizationTo maintain the structure and integrity of a protein, it is essential to prevent degradation oraggregation. This can be achieved through the addition of protease and phosphatase inhibitors tobuffers and solutions. Widely cited, cOmplete and PhosSTOP protease and phosphatase inhibitorsfrom Roche are easy-to-use, flexible tablet-based formulations which inhibit a wide range of proteasesand phosphatases respectively.cOmplete Protease Inhibitor CocktailEach of these convenient water-soluble tablets containsa broad blend of metalloproteases, serine, and cysteineprotease inhibitors to ensure the protection of proteinsisolated from almost any tissue or cell. This includesmaterial derived from animals, plants, yeast, bacteriaand fungi. Provided as a formulation containing EDTA,as well as EDTA-free tablets to leave the stability andfunction of metal-dependent proteins unaffected,cOmplete Protease Inhibitor Cocktails require noweighing or measuring and dissolve rapidly in solution. Easy to use Protection against a broad range of proteases Suitable for extracts derived from almost any tissueor cell10The Complete Roche PortfolioPhosSTOP Phosphatase Inhibitor CocktailProvided as a quick-dissolving, water-solubleformulation, PhosSTOP Phosphatase Inhibitor Cocktailtablets contain a proprietary blend of acid and alkalinephosphatase inhibitors, serine/threonine phosphataseinhibitors and tyrosine protein phosphatase inhibitors.Effective across a wide range of sample materials,including tissues and cells of mammalian, plant,yeast or bacterial origin, PhosSTOP PhosphataseInhibitor Cocktail tablets can be easily combinedwith cOmplete Protease Inhibitor Cocktail forcomprehensive protein protection. Easy to use Protection against a broad range of phosphatases Easily combined with cOmplete Protease InhibitorCocktailCat. No.DescriptionCO-ROcOmplete Protease Inhibitor CocktailPHOSS-ROPhosSTOP Phosphatase Inhibitor Cocktail

Cellular AnalysisTransfectionInvolving the introduction of DNA or RNA into eukaryotic cells, transfection is a widely used techniquefor the study and modulation of gene expression. During stable transfection, the genetic material isincorporated into the genome of the recipient; whereas, during transient transfection expression ofthis material is relatively short-lived. Various transfection methods have been developed, includingphysical, chemical and biological techniques.X-tremeGENE Transfection ReagentsCreated to afford high transfection efficiency, ease-ofuse and reproducibility, X-tremeGENE TransfectionReagents provide more viable cells and morephysiologically relevant results. Including productsoptimized for use with established cell lines, primarycells, stem cells and insect cell lines, as well asX-tremeGENE siRNA Transfection Reagent to knockdown gene expression in many different cell types, theX-tremeGENE product range demonstrates activityin the presence of serum to avoid the need for mediachanges. Quick and easy to use Low cytotoxicity Minimal optimization requiredX-tremeGENE Transfection Reagents Performance ytotoxicity (%)7030Luciferase (RLU)% of transfected PXH2:1XHPXHPneg0Figure 3: X-tremeGENE Transfection Reagents Performance.(A) X-tremeGENE HP (XHP) and X-tremeGENE 9 (X9) transfectionefficiency compared to LTX (μL reagent : μg plasmid DNA ratios).(B) A549 cells were transfected with luciferase encoding plasmid DNAusing either X-tremeGENE 360 (XTG360), Lipofectamine 2000(Thermo Fisher Scientific) or Lipofectamine 3000 (Thermo Fisher010Cellsalone2:13:14:1X-tremeGENE 3603:14:15:1Lipofectamine 20001.5:2:12:2:13:2:10Lipofectamine 3000Scientific) at indicated reagent-to-DNA ratios or reagent-to-P3000 to-DNA ratio. Higher transgene expression (luciferase) and lowercytotoxicity was observed in cells transfected with XTG360 at optimalratios compared to cells transfected with Lipofectamine 2000 orLipofectamine 3000.11

X-tremeGENE 9 Transfection ReagentX-tremeGENE 360 Transfection ReagentSuitable for commonly used cell lines, theX-tremeGENE 9 transfection reagent providesmaximal cellular viability post-transfection forphysiologically relevant results. Transfection with low cytotoxicity Proprietary blend of lipids designed to reduce timeconsuming transfection optimization Suitable for use with or without serumX-tremeGENE HP Transfection ReagentProviding reliable transfection results for hard-totransfect cell lines, the X-tremeGENE HP transfectionreagent is a multi-component reagent that is suitablefor insect or animal cells. Free of animal-derived components Suitable for cancer and primary cell lines Low cytotoxicity facilitates generating physiologicallyrelevant dataX-tremeGENE transfection reagents are characterizedby their exceptional performance in delivering maximaltransfection efficiencies while minimizing cytotoxicity.The X-tremeGENE 360 transfection reagent isoptimized for use with common and hard-to-transfectcell lines and a variety of applications and molecules,including plasmid DNA, siRNA/miRNA, and Cas9/gRNA RNP. Simplify your molecular workflow and gainconfidence with the X-tremeGENE 360 transfectionreagent for your gene expression and genome editingapplication needs. Utilizes a next-generation synthetic polymerformulation to maximize transfection efficiency Provides reduced cytotoxicity compared to traditionalcationic lipid transfection reagents Free of animal-derived componentsCat. No.DescriptionXTG9-ROX-tremeGENE 9 DNA Transfection ReagentXTGHP-ROX-tremeGENE HP DNA Transfection ReagentSITRAN-ROX-tremeGENE siRNA Transfection ReagentXTG360-ROX-tremeGENE 360 Transfection ReagentFor more information, visitSigmaAldrich.com/xtremegene12The Complete Roche Portfolio

Cell IsolationWhile many techniques are used to generate data from heterogeneous cell populations, it is often moreinformative to separate and analyze individual cellular sub-types. By isolating specific cells and growinghomogeneous populations in culture, it is possible to study unique cellular signaling pathways ordiffering developmental behaviors, and to detect genotypic or phenotypic features that may otherwisebecome overlooked.CollagenaseLiberase Research Grade EnzymesCollagenase is widely used for the disaggregationof tissues and for generating single cell suspensionsin order to establish primary cell cultures. Preparedfrom Clostridium histolyticum cultures by filtration,ammonium sulfate precipitation, dialysis, andlyophilization, collagenase products from Roche havebeen cited for the preparation of cell types whichinclude hepatocytes, adipocytes, pancreatic islets,epithelial cells, muscle cells and endothelial cells. Specifically degrades native collagen Uninhibited by serum Convenient, lyophilized formulationContaining highly purified Collagenase I andCollagenase II, Liberase Research Grade enzymesoffer significantly higher specific activities thantraditional collagenases due to enhanced purity of thecollagenase enzymes. The enzyme blend also offersthe advantage of lower clostripain and trypsin activityas compared to traditional collagenase, in addition toreduced endotoxin content. Suitable for the dissociationof a broad range of tissue types, the high purity ofLiberase Research Grade enzymes contributes to highcell yield and viability. Maximize viability and yield of isolated cells Highly pure enzymes afford superior specific activity Free of mammalian or avian tissue-derived rawmaterialsCat. No.DescriptionCOLLD-ROCollagenase DCOLLA-ROCollagenase ACOLLP-ROCollagenase PCOLLDISP-ROCollagenase/Dispase5401020001Liberase TL Research GradeLIBTH-ROLiberase TH Research GradeLIBTM-ROLiberase TM Research GradeLIBDL-ROLiberase DL Research GradeLIBDH-ROLiberase DH Research Grade13

Cell FunctionWhile different cell types perform a vast range of diverse functions, proliferation and death areprocesses common to a large assortment of cell types. Measurement of these responses can be highlyinformative, for example providing an indication of cell health and viability or suggesting whethera potential drug candidate may be cytotoxic. The Roche biochemical reagents portfolio includesoptimized ELISA kits for the measurement of cellular proliferation and cell death.Cell Proliferation ELISA, BrdU (colorimetric)In Situ Cell Death Detection Kit, FluoresceinA colorimetric immunoassay based on themeasurement of BrdU incorporation during DNAsynthesis, the Cell Proliferation ELISA is a precise, rapidand simple assay suitable for use in in many differentin vitro cell systems. Specificity is assured throughnon-reactivity of the peroxidase-conjugated anti-BrdUantibody with endogenous cellular components such asthymidine, uridine or DNA. Second generation kit with colorimetric quantificationof cell proliferation Identifies BrdU-labeled denatured DNA Increased safety with no use of radioactive[3H]-thymidine

KAPA2G Fast HotStart ReadyMix Containing the engineered KAPA2G Fast HotStart DNA Polymerase, KAPA2G Fast HotStart ReadyMix reduces cycling times by up to 75%. This is achieved using 1 second extension times for amplicons 1 kb (15 sec/kb for longer amplicon