Follow the protocol in the user manual for your specific smart cdna based application. This robust reverse transcription kit allows a selection of firststrand priming strategies, oligodt primers only, random primers only, or userdesigned genespecific primers. Proceed to first strand cdna synthesis first strand cdna synthesis 1. The optimal quantity of rna template depends on the rna purity and the expression level of the particular mirna of interest. Protocols cdna synthesis from total rna or mrna since the conditions described for the first strand reaction are optimized, do not change volumes and concentrations. Firststrand cdna synthesis incorporating dutp and 5.
The kit uses revertaid reverse transcriptase rt, a recombinant mmulv rt which maintains activity at 4250c and is suitable for synthesis of cdna up to kb. If the firststrand cdna synthesis 33 dnarna hybrid itself is the starting material for secondstrand synthe sis this volume 35. For robust twostep rtpcr and rtqpcr of rna up to 14 kb. Superscript iii firststrand synthesis system for rtpcr. The superscript iv firststrand synthesis system contains all components needed for rt reactions, plus an additional control gene and primers, and provides the flexibility to customize the rt setup to fit the needs of your application. Norgens truscript first strand cdna synthesis kit for mrna is an allinone. Figure 2 shows that cdna prepared with the marligens firststrand cdna synthesis system provides significantly enhanced sensitivity compared to cdna prepared with a competitors kit. Thermo scientific revertaid first strand cdna synthesis kit is a complete system for efficient synthesis of first strand cdna from rna templates.
For the first strand synthesis of singlestranded cdna from rna for use as a pcr template. A control reaction without reverse transcriptase is recommended to examine the dna contamination in the samples. Invitrogen superscript iii firststrand synthesis supermix quanta qscript cdna synthesis kit quanta qscript cdna supermix. Accuscript high fidelity 1st strand cdna synthesis kit. If the synthesis of secondstrand cdna is contemplated by the classi cal method this volume 34, rna must be hydrolyzed step 3. For this purpose the specialized enzyme reverse transcriptase is utilized, which converts singlestranded rna ssrna into dna. Truscript first strand cdna synthesis kit product insert. Hyperscripttm first strand synthesis kit is ideally organized for synthesizing reaction of first strand cdna from purified mrna or total rna. Reverse transcriptase and either random hexameric primers or an oligodt. This is recommended when the cdna synthesis reaction uses less than 10 ng of rna input. It uses cloned moloney murine leukemia virus mmlv reverse transcriptase to synthesize first strand cdna from an mrna population. Superscript iii firststrand synthesis supermix for qrtpcr. Amplification of firststrand cdna using the polymerase chain reaction.
Pdf extralong firststrand cdna synthesis researchgate. Assay sensitivity and dynamic range various amounts of hela cell total rna, ranging from 850 ng down to 10 pg, were reverse transcribed using anchored oligodt 23 vn as. Using the fermentas first strand cdna synthesis kit quantify we need 0. These are available as convenient pdf files online at. Firststrand cdna synthesis was performed using a modified dntp mixture that contained dutp and a mmlv reverse transcriptase. The iscript select cdna synthesis kit is a sensitive, flexible, and easytouse kit for the generation of firststrand cdna. The superscript iv synthesis system is the top choice for performance and flexibility for rtpcr applications. Procedure for firststrand cdna synthesis norgen biotek corp. Dear clay dont worry about it, just follow the protocol, only after cdna synthesis, plz dilute the cdna 3 to 5 times with nuclease free water and then use it as template for qpcr, u will be doing. The sonication step for shearing doublestranded cdna may lead to the trimming of both linear and hairpinstructured 5. Use of manganese chloride manganese chloride is provided in the mirna 1ststrand cdna synthesis. Rna isolationpurification and firststrand cdna synthesis.
This kit is powered by primescript reverse transcriptase primescript rt, which has exceptionally strong stranddisplacement activity and efficiently synthesizes cdna. The unlabeled crna is then reverse transcribed in the firststrand cdna synthesis step of the second cycle using random primers. Truscript first strand cdna synthesis kit for mrna product. Kits varied in their ability to efficiently generate cdna from different rna inputs linearity of cdna synthesis kits with bactin 15 20 25 30 35 401. Firststrand synthesis is driven by amv avian myeloblastosis virus. First strand cdna synthesis using the neb protoscript kit adapted from neb protocol overview reverse transcriptase is an enzyme capable of synthesizing a strand of dna the first strand complementary to the mrna template cdna. Mix and spin each component in a microcentrifuge before use. Transcriptor reverse transcriptase the core component of the kit is a recombinant reverse transcriptase expressed in e. Complementary dna an overview sciencedirect topics.
Invitrogen superscript iv firststrand synthesis system. Readytogo youprime firststrand beads, prep aration and setup are convenient. Reverse transcriptases rts use an rna template and a short primer complementary to the 3 end of the rna to direct the synthesis of the first strand cdna, which can be used directly as a template for the polymerase chain reaction pcr. Priming using random primers is the most generally used method of initiating cdna synthesis from a variety of rna templates.
A protocol for amplification using stratagene highfidelity. Sequencing of firststrand cdna library reveals fulllength transcriptomes. First strand cdna synthesis protocols e6300 introduction. Increase the temperature of first strand reaction up to 55c. For example, 10 minutes incubation can be used for up to 5 kb cdna synthesis. Instruction manual protoscript ii first strand cdna. The cdna reaction product can be used directly in the next step without being purified. Goscript reverse transcriptase reverse transcription. However, amplification of some pcr targets 1 kb may require the removal of rna complementary to the cdna. Firststrand cdna synthesis for quantitative rtpcr origene.
All subsequent steps require that the synthesis step produce cdna with high fidelity, accurately representing the target dna. Do not proceed with the protocol until a primer for reverse transcription has been selected. The resulting singlestranded cdna is suitable for use in realtime quantitative reverse transcription pcr rtqpcr. Superscript iii firststrand synthesis system for rtpcr author. First strand cdna synthesis after thawing, mix and briefly centrifuge the components of the kit. Is cdna amplification recommended after first strand cdna. Studying gene expression levels, via twostep rtpcr, using qualitative rtpcr or quantitative rtpcr on the lightcycler carouselbased system, the lightcycler 480 system, or other real. Protocol for cdna synthesis and qrtpcr cdna synthesis. The completed firststrand reaction is then available for pcr amplification or. Pdf on jun 1, 2002, p carninci and others published extralong firststrand cdna synthesis find, read and cite all the research you need on.
Roche diagnostics transcriptor first strand cdna synthesis. For the most uptodate protocols, please visit our website at iv. Nzy firststrand cdna synthesis kit is formulated to provide high yields of fulllength cdna products and to increase. Nzy firststrand cdna synthesis kit is a system that includes all the necessary components to synthesize firststrand cdna, except the template rna. Accuscript high fidelity 1st strand cdna synthesis kit agilent. Sequencing of firststrand cdna library reveals full. Usb firststrand cdna synthesis kit for realtime pcr. This kit provides all components for cdna synthesis, including reverse transcriptase, rnase inhibitor, oligo dt, random hexamer, dntps and nuclease free water. A laboratory manual, 2nd edition, cold spring harbor. The transcriptor first strand cdna synthesis kit is designed to reverse transcribe rna mrna, total rna, viral rna, and in vitrotranscribed rna from a variety of sources for the following applications. Rna isolationpurification and firststrand cdna synthesis entered by kevin janes janes lab protocols 122816 2 wet pellets can be warmed for a few seconds at 37oc to increase evaporation smaller or broken pellets will need to have the remaining liquid evaporated off at 42. Reverse transcriptases rts use an rna template and a primer complementary to the rna to direct the synthesis of the first strand cdna, which can be used directly as a template for the polymerase chain reaction pcr.
Figure 1 is a summary of the two procedures that may be. The synthesis of dna from an rna template, via reverse transcription, produces complementary dna cdna. Nzy firststrand cdna synthesis kit cdna kits nzytech. Roche diagnostics transcriptor first strand cdna synthesis kit, 100 reactions. The first is the difficulty in reaching the cap site in the firststrand synthesis with the reverse transcriptase rt, the first enzyme involved in the preparation of a cdna library. Superscript iii firststrand synthesis supermix for qrtpcr provides the hightemperature capability of superscript iii reverse transcriptase in an optimized supermix format for the synthesis of firststrand cdna for use in realtime quantitative rtpcr qrtpcr. Thaw all necessary components and place them on ice. Mix rna sample and primer dt 23 vn in two sterile rnasefree microfuge tubes. In this experiment, cdnas prepared using each method were amplified in real time with primers specific for the 5 end of the adar gene using three different. Universal ribocloner cdna synthesis system technical manual. Hyperscript first strand synthesis kit 1 bohemia genetics. Synthesis kit is a complete system for efficient synthesis of first strand cdna from mrna or total rna templates. Firststrand cdna synthesis using superscript ii rt, continued the cdna can now be used as a template for amplification in pcr.