Template and primer requirements for reverse transcriptase 3. Arts journalism and criticism in a digital age, a threeday conference of panels, keynotes, and a blog mentorship program, all dedicated. Invitrogen superscript iv reverse transcriptase fisher. The reverse transcription yields for the rna multistandard varied more than 100fold. Mixtures of all reaction components, except for reverse transcriptase, were held at different temperatures for 3 min. Description superscript iv reverse transcriptase rt is significantly improved over superscript iii in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of the previous enzyme, including increased thermostability, highly efficient fulllength cdna synthesis, and reduced rnase activity. Invitrogen superscript ii reverse transcriptase 1st strand. Superscript iii platinum twostep qrtpcr kit for lux and probe based detection systems superscript iii platinum twostep qrtpcr kit with sybr green for detection based on sybr green kit components. To one reaction add 1 ml superscript ii reverse transcriptase. The iscript reverse transcription supermix for rtqpcr is a simple, fast, and sensitive firststrand cdna synthesis kit for gene expression analysis using realtime qpcr. Superscript iv reverse transcriptase rt is significantly improved over superscript iii in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of the previous enzyme. This enzyme is efficient in detecting rna from samples containing inhibitors, such as alcohols, cell lysis reagents, salts, and those inherent in biological samples, such as those found in blood, feces, and ffpe rna. Rt binds not only nucleic acid duplexes, but also singlestranded rna 29, and this may explain why the introduction of nonhomologous rna relieves the inhibition for this particular type of rt 1, but it has no effect on mmlv, h.
The use of superscript iii reverse transcriptase ensures high specificity and high yields of cdna from small amounts of starting materialas little as 10 pg total rna. It is significantly improved over superscript iii in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of the previous enzyme, including increased thermostability, highly efficient fulllength cdna synthesis, and reduced. But i was using invitrogen superscript ii as well iii reverse transcriptase kit for cdna synthesis. Comparison of reverse transcriptases in gene expression. Reverse transcription of sodium borohydridetreated ac4c results in partial incorporation of adenosine in the cdna strand opposite the reduced ac4c. Superscript iii superscript iii reverse transcriptase rnase hm mlv rt cdna. Reverse transcription an overview sciencedirect topics. This enzyme is efficient in detecting rna from samples containing inhibitors, such as alcohols, cell lysis reagents. Superscript iii reverse transcriptase is an engineered version of mmlv. Reverse transcriptases are used by retroviruses to. The latter was overall the most efficient reverse transcriptase, with a mean yield of 83%. Rna with 200 units of cyclescript reverse transcriptase for 3 hours at 37.
Superscript iv reverse transcriptase for rna analysis. Reverse transcriptase with stability at higher reaction temperatures. Testing gene expression by reverse transcriptase pcr rt. The reverse transcriptase from wt moloney murine leukemia virus mulv, the parent of superscript iii, plus the rts from avian myeloblastosis virus amv and from hiv1 and hiv2 were. Crossclade simultaneous hiv drug resistance genotyping. Reverse transcriptases are used by retroviruses to replicate their genomes, by retrotransposon mobile genetic elements to proliferate within the host genome, by eukaryotic cells to extend the telomeres at the ends of their linear chromosomes, and by. Invitrogen superscript iv reverse transcriptase 2,000. Description superscript iv reverse transcriptase rt is significantly improved over superscript iii in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of the previous. The use of cloned moloney murine leukemia virus reverse transcriptase to synthesize dna from rna. Invitrogen superscript iv reverse transcriptase 2,000 units. Superscriptz iii reverse transcriptase is a version of mmlv rt that has been engineered to reduce rnase h activity and provide increased thermal stability 1, 2.
In one tube, the preblended 5x supermix contains all the necessary components except rna template for reverse transcription. The enzyme can synthesize cdna at a temperature range of 45. Fluorescencebased reverse transcription, realtime quantitative pcr rtqpcr is a highly sensitive method for quantification of specific rnas, whether cellular e. Goscript reverse transcriptase reverse transcription cdna. Invitrogen superscript ii reverse transcriptase fisher. This enzyme is genetically engineered by the introduction of point mutations in the rnase h active center to reduce rnase h activity. This is the main change between the two invitrogen products, and according to the manufacturer, this increased temperature leads to more efficient reversetranscription due to the reduced presence of rna. Invitrogen superscript iii reverse transcriptase 200. Testing gene expression by reverse transcriptase pcr rtpcr. Superscript ii reverse transcriptase rt is a dna polymerase that synthesizes a complementary dna strand from singlestranded rna, dna, or from an rna. Am9738 and reverse transcribed using oligodt primer and goscript or superscript iii reverse transcriptase, following the manufacturers instructions with the exception that all reactions were carried out at 50c. After synthesis, the firststrand cdna can be amplified with specific primers by pcr without intermediate organic extractions or ethanol precipitations. Invitrogen superscript iii reverse transcriptase 50. Goscript reverse transcriptase reverse transcription.
Superscript iv reverse transcriptase protocol outline a. Add reaction mix to annealed rna rt reaction setup use the measurements below to prepare. The recombinant moloney murine leukemia virus reverse transcriptase rt. For example, the separate reverse transcription step delays the results by up to 30 min, low temperature dnadependent dna polymerase activity of the reverse transcriptase can catalyze non. In the absence of rnase h, switching still occurred but the efficiency was lowered. Reverse transcription is performed using superscript iii reverse transcriptase invitrogen using the suppliers instructions except that the reaction is carried out for 1h at 50 to disrupt secondary structures in mrna. Variables influencing the efficiency and interpretation of reverse. Label this tube rt incubate the reactions in a water bath at 42oc for 50 minutes. Pdf has a text raise command ts, which can be used to make a superscript, but it doesnt change font size, so that should be done manually. It can finalize firststrand cdna synthesis in just 30 minutes, which reduces the total time required for rt. Reverse transcription using superscript iii and oligodt1218 first strand cdna synthesis from rna 1. The enzyme is used to synthesize cdna at a temperature range of 4255c, providing increased specificity, higher yields of cdna, and more fulllength product than other reverse transcriptases. A reverse transcriptase rt is an enzyme used to generate complementary dna cdna from an rna template, a process termed reverse transcription. Superscript iii reverse transcriptase thermo fisher scientific.
In may 2015, the walker art center and mn artists hosted superscript. Superscript iv reverse transcriptase rt is significantly improved over superscript iii in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of the previous enzyme, including increased thermostability, highly efficient fulllength cdna synthesis, and reduced rnase activity. Jun 08, 2015 to meet this performance gap, the thermo fisher life sciences solutions group produced a new reverse transcriptase, superscript iv, and experiments we performed show that it is the most robust. Goscript reverse transcriptase utilizes mmlv and stateoftheart buffer technology designed for. Stimulation of reverse transcriptase generated cdnas with. Superscript iii reverse transcriptase rt is active at 50c and has a halflife of 220 minutes, providing increased specificity with genespecific primers gsps and the highest cdna yield of all rts. Cyclescript reverse transcriptase is engineered for improved performance over a broad range of cdna synthesis synthesis temperature from 37 to 55 it is exceptionally unique product having superior. Transcriptor reverse transcriptase is a fast, recombinant reverse transcriptase expressed in e. Superscript iv reverse transcriptase for rna analysis eshg. Helpful for preparing long cdnas and libraries with a high percentage of fulllength. This optimal reaction temperature differs from superscript iiis predecessor, superscript ii, which showed optimal activity at 42. Invitrogen superscript iv reverse transcriptase is the latest superscript enzyme, engineered to deliver superior performance even with challenging rna samples. It is a dna polymerase that synthesizes a complementary dna strand from singlestranded rna dna or an rna. Label this tube rt incubate the reactions in a water bath at 42oc for 50 minutes, then in a heat block at 85oc for 5 minutes.
Superscript iii reverse transcriptase is a version of mmlv rt that has been engineered to reduce rnase h activity and provide increased thermal stability. Reverse transcription using superscript iii and oligodt. It should also be noted that reduced ac4c results in. Mmlv reverse transcriptase is isolated from a li strain containing a recombinant clone. Invitrogen superscript iii reverse transcriptase is a genetically engineered mmlv reverse transcriptase rt that was created by introduction of several. Pdf template switching by reverse transcriptase during. Wed like to understand how you use our websites in order to improve them. Goscript reverse transcriptase overview advantages ordering information goscript reverse transcriptase is optimized for qpcr assays and delivers robust, reliable cdna synthesis. Reverse transcriptase, also called rnadirected dna polymerase, an enzyme encoded from the genetic material of retroviruses that catalyzes the transcription of retrovirus rna ribonucleic acid into dna.
Other factors required for reverse transcription 3. Because superscript iii rt is not significantly inhibited by. Goscript reverse transcriptase overview ordering information long transcripts secondary structure inhibitor resistance activitycost effectiveness advantages sensitivity goscript shows equal or greater sensitivity at all transcript levels 15 20 25 30 35 40 sensiscript omniscript superscript iii superscript ii goscript 10,000. Messenger rna profiling relies heavily on a reverse transcription step which is accompanied by pcr. Crossclade simultaneous hiv drug resistance genotyping for. Superscript iii firststrand synthesis system for rtpcr. Superscript ii rnase h reverse transcriptase from invitrogen. For cdna synthesis with integrated removal of genomic dna contamination for use in realtime twostep rtpcr. Rt with reduced rnase h activity and increased thermal stability.
Pcr inhibition by reverse transcriptase leads to an. Reverse transcription using superscript iii and oligodt1218. Jun 06, 2015 the superscriptiv reverse transcriptase is an example of such a reagent. Add reaction mix to annealed rna rt reaction setup use the measurements below to prepare your rt reaction, or enter your own parameters in the column provided. Superscript iii reverse transcriptase 2,000 units total, at 200ul is supplied with a vial 1 ml of 5x firststrand buffer 250 mm trishcl ph 8. Superscript iii firststrand synthesis system for rtpcr author. The basic reaction, with rna templates and normal reverse transcriptase, yielded as much as 83% template switching. Protoscript ii reverse transcriptase new england biolabs. Pdf reverse transcriptase inhibits taq dna polymerase.
Natural primers for cdna synthesis of retrovirus genome 3. The enzyme is used to synthesize cdna at a temperature. In this study, we used purified bacteriophage ms2 genomic rna as a model virus target to test two different rt enzymes superscript ii and superscript iii, two. Sep 01, 2004 the reverse transcription yields for the rna multistandard varied more than 100fold. Invitrogen superscript iii reverse transcriptase 50 reactions.
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 reverse transcriptase from invitrogen. Superscript iii reverse transcriptase is a version of m mlv rt that has been. Reverse transcriptase activity in other organisms 4. Viral rna was subjected to onestep rtpcr amplification using superscript iii reverse transcriptase and high fidelity platinum taq polymerase invitrogen and the primers hiv2252f and. The reverse transcriptase from wt moloney murine leukemia virus mulv, the parent of superscript iii, plus the rts from avian myeloblastosis virus amv and from hiv1 and hiv2 were similarly tested with wtsla7, and mutsla7 under the nine dntps concentration conditions. Superscript iii reverse transcriptase 10,000 units total, at 200ul is supplied with a vial 1 ml of 5x firststrand buffer 250 mm trishcl ph 8. Superscript reverse transcriptases thermo fisher scientific. This is the main change between the two invitrogen products, and. Nov 27, 2005 the halflife of superscript iii is 220 min versus 6 min for superscript ii at 50c. Superscript iii onestep rtpcr system with platinum taq.
Pdf reverse transcriptase inhibits taq dna polymerase activity. There is also a baselineshift property for texts that can be used. Component concentration amount added oligodt 1218 500ng 200500ng 1. Dec 23, 2014 viral rna was subjected to onestep rtpcr amplification using superscript iii reverse transcriptase and high fidelity platinum taq polymerase invitrogen and the primers hiv2252f and hiv5073r. Superscript iv reverse transcriptase rt is a proprietary mmlv mutant with superior robustness and reliability in rt reactions. Basically, if i have 1 ug of rna, i used 1 ul of enzyme and if it is less than that for eg.