Use random hexamers instead of oligodt in the firststrand reaction. Synergistic actions of blocking angiopoietin2 and tumor. You can use as little as 100ng total rna and still detect you gene expression in pcr. Upscaling cdna synthesis using superscript iii reverse transcriptase posted in pcr, rtpcr and realtime pcr. Superscript iii firststrand synthesis supermix for qrtpcr provides the high temperature capability of superscript iii reverse transcriptase in an optimized. Transscript firststrand cdna synthesis supermix with. For cdna synthesis i used smart first strand cdna synthesis kit, eurx cat. Upscaling cdna synthesis using superscript iii reverse. Superscript iii platinum twostep qrtpcr kit with sybr green. Abi prism 7000 sequence detection system applied biosystems. Description using the superscript iii firststrand system. Invitrogen superscript iv firststrand synthesis system. Superscript iii firststrand synthesis supermix thermo fisher.
Oligodt 20 50 m, 50 l random hexamers 50 ngl, 250 l 10x rt buffer, 1 ml 0. The amount of rna needed for superscript iii firststrand. Superscript iii firststrand synthesis supermix for qrtpcr provides the hightemperature capability of superscript iii reverse transcriptase in an optimized. Rnacdna superscript iii firststrand synthesis system for rtpcr. Pcr results conducted in thermal cycler with both conventional pcr and with evagreen mix. The cdna is efficiently synthesized by transscript rtri enzyme mix and 2x ts reaction mix. In the second step, pcr is performed in a separate tube using primers specific for the gene. Advantages of the transscript firststrand cdna synthesis supermix deficient rnase h activity to reduce rna template. Kit includes superscript iiirnaseout enzyme mix, 2x firststrand reaction mix, and annealing buffer superscript iii reverse transcriptase, included in the enzyme mix, is a version of mmlv rt that has been engineered to reduce rnase h activity and provide increased thermal stability. Invitrogen superscript iii firststrand synthesis supermix 50. Superscript iii firststrand synthesis supermix thermo.
Troubleshooting sybr greener qpcr supermix universal. Readyscript cdna synthesis mix complete reagent for first strand. Mmlv reverse transcriptase, rnase h minus, point mutant. Reagents are provided for 50 or 250 rt reactions of 20 l each. Sybr green pcr master mix, 200 reactions applied biosystems. Simplified rt reverse transcription animation youtube. It is a dna polymerase that synthesizes a complementary dna strand from singlestranded rna dna or an rna.
Invitrogen superscript iii firststrand synthesis system. For multiple reactions, prepare a master mix of components common. Readyscript cdna synthesis mix is a sensitive and easytouse solution for twostep rtpcr. Superscript ii rnase h reverse transcriptase invitrogen, cat. The superscript iv first strand 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. Rna purity and concentration were determined spectrophotometrically.
The enzyme is widely used to synthesize firststrand cdna at. Superscript iii firststrand synthesis system for rtpcr author. The superscript iii firststrand synthesis system for rtpcr is supplied with the following. Superscript iv first strand synthesis kit invitrogenis. Total rna 5 g random hexamers 50 ngl 3 l 10 mm dntp mix 1 l depc h2o to 10 l 2. Superscript firststrand synthesis system for rtpcr invitrogen. Enzyme mix, reaction mix, rna template 25 degrees 10 42 degrees 50 70 degrees 20 add 1ul rnase hreaction 37. The system uses a mixture of superscript iii reverse transcriptase and platinum taq high fidelity dna polymerase for enhanced rtpcr yields and fidelity, as well as the detection of longer templates.
For the twostep method, the first strand cdna synthesis was created in a total volume of 40. Superscript iii firststrand synthesis system from invitrogen. Superscript iii firststrand synthesis system for rtpcr. Invitrogen superscript iii firststrand synthesis system 50. Prepare the following rnaprimer mixture in each tube. Add 1 l 200 units of superscript iii reverse transcriptase and mix gently by pipetting up and down. In one tube, the preblended 5x supermix contains all the necessary components except rna template for reverse transcription. How do i choose between lunascript rt supermix kit and protoscript first strand cdna synthesis kits. I am using superscript iv first strand synthesis kit for making cdna from rna samples. Invitrogen superscript iii firststrand synthesis supermix for. Since random hexamers have shown to result in an overestimation of mrna copy number by up to 19fold 50, we used oligodt primers instead. An article comparing mmlv reverse transcriptase, rnase h minus, point mutant from promega, which has similar thermal stability properties, produces long cdna transcripts and performs similarly in rtpcr, to superscript ii, making it a good alternative reverse transcriptase. If you are using oligodt1218 or gsp, incubate at 42 c for 2 mins. For the cdna synthesis, i am going to use superscript iii rt with oligo dts.
Hi all,i am planning to do rtpcr on rna samples 5ug which are quite precious because they are not mine, they were a gift. So in case of 20ul reaction that means only 3 different genes in triplicates 2 ul in triplicates, i. It also has increased thermal stability and so is effective up to 50. I use this kit routinely to synthesize cdna from 100 ng1 ug of rat heart total rna. Provides the hightemperature capability of superscript iii reverse transcriptase in an optimized supermix format for synthesis of firststrand cdna for use in. Superscript ii rnase h reverse transcriptase from invitrogen,superscript ii rnase h reverse transcriptase rt is an improved version of the superscript rnase h rt. Superscript iii firststrand synthesis supermix for qrtpcr. Here is a summary of our three first strand cdna synthesis kits.
Superscript iii onestep rtpcr system with platinum taq. The superscript iv synthesis system is the top choice for performance and flexibility for rtpcr applications. Efficient recovery of whole blood rna a comparison of. Reagents are provided for 50 or 250 rt reactions of 20l each. How do i choose between lunascript rt supermix kit and. Superscript firststrand synthesis system for rtpcr from.
This method of first strand cdna synthesis is more efficient than readytogo beads. Reverse transcription is carried out with the superscript firststrand synthesis system for rtpcr. It has a lower rnase h activity and a high thermal stability. Choose this cdna synthesis kit when you want to synthesize fulllength cdna with provided primers select from oligo dt primer or random 6mers, both. Readyscript cdna synthesis mix is a sensitive and easytouse solution for two step rtpcr.
Maintain an elevated temperature after the annealing step, as described in the protocol for cdna synthesis from highgc content transcripts, page 3. I prime my firststrand cdna synthesis reaction using the oligodt provided in the kit, which hybridizes to 3. The first strand cdna product generated is up to 10 kb. Now transgen is a leading manufacturer of more than 200 molecular and cellular biology reagents. Using superscript iii firststrand synthesis supermix this supermix formulation can be used to quantify fewer than 10 copies of a target gene in qrtpcr, with a broad dynamic range that supports accurate quantification of highcopy mrna from up to 1 g of total rna. Services offered milliq services lanexo system emprove program basic research product. Superscript ii rnase h reverse transcriptase from invitrogen.
Rna targets from 100 bp to 12 kb can be detected with this system. This 5x concentrated master mix provides all necessary components except rna template for firststrand synthesis including. The rt conditions were the same as onestep except that a 10min incubation at 25c was included at the start due to the use of a mix of random hexamersoligo dt primers and a 5min incubation at 85c was included at the end to terminate the. The invitrogen superscript iii firststrand synthesis supermix for qrt pcr 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. Superscript iii firststrand synthesis system for rtpcr cat. Invitrogen superscript iii firststrand synthesis supermix. Increase the temperature of firststrand reaction up to 55c. Mix contents of the tube gently by pipetting up and down. If we need to run more genes, we increase the amount of rna put into rt transcriptor limit is 5 ug and then dilute the rt mix. Because superscript iii rt is not significantly inhibited by ribosomal and transfer rna, it can be used to synthesize cdna from total rna. The 260280 and 260230 values of my rna samples are above 2. The amount of starting material can vary from 1 pg5 g of total rna.
The following procedure is based on invitrogens protocol. Analysis of onestep and twostep realtime rtpcr using. For the pcr reaction, one of the following dna polymerases is recommended. Superscript iii onestep rtpcr system with platinum taq high. Invitrogen superscript iii firststrand synthesis system 50 reactions. Problem possible cause solution platinum sybr green qpcr. Mix gently 42 degrees for 2 add 1ul ssiiireaction 25 degrees 10 42 degrees 50 85 degrees 5 add 1ul rnase hreaction 37 degrees 20 htp protocolmastermix format combine components for firststrand synthesis. Complete reagent for first strand cdna synthesis for rtqpcr. Dec 06, 2012 reverse transcription is a widely used technique in molecular biology where a rna strand is reverse transcribed into its dna complement using the reverse transcriptase, such as superscript.
Protoscript ii first strand cdna synthesis kit neb. Invitrogen superscript iii firststrand synthesis system 50 reactions life. Bring 2 g total rna in the volume of 20 l with rnase free water. The iscript reverse transcription supermix for rtqpcr reaction setup for a single cdna synthesis reaction iscript rt supermix is a sensitive, fast, and convenient reagent for gene expression analysis using realtime reverse transcription quantitative pcr rtqpcr and standard rtpcr. Protocol for cdna synthesis and qrtpcr cdna synthesis. Superscript iv reverse transcriptase user guide 3 february 201526 superscript iv firststrand cdna synthesis reaction the example procedure below shows appropriate volumes for a single 20l reverse transcription reaction. This kit is an optimized master mix, including a blue visible tracking dye, for first strand cdna synthesis for use in a twostep rtqpcr workflow.
The 2x rt reaction mix includes oligodt 20, random hexamers, mgcl 2, and dntps in a buffer formulation that has been optimized for firststrand synthesis of cdna for use in realtime rtpcr. The first strand cdna synthesis kits contain different reverse transcriptases with the major differences being their primary applications, format, and reaction temperature. Invitrogen superscript iii firststrand synthesis supermix 50 reactions life sciences. Sep 19, 2006 superscript ii rt is not inhibited by ribosomal rna and transfer rna, so it can effectively synthesize firststrand cdna from a total rna preparation. Using superscript iii firststrand synthesis supermix this supermix formulation can be used to quantify fewer than 10 copies of a target gene in qrtpcr, with a broad dynamic range that supports accurate quantification of highcopy mrna from up to 1g of total rna. The iscript reverse transcription supermix for rtqpcr is a simple, fast, and sensitive first strand cdna synthesis kit for gene expression analysis using realtime qpcr. Transscript firststrand cdna synthesis supermix transscript firststrand cdna synthesis supermix provides all the necessary components for cdna synthesis from total rna or mrna. The firststrand cdna obtained in the synthesis reaction may be used directly to amplify target cdna by pcr or stored at 20.
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