cdna synthesis protocol thermo scientific

165 0 obj << /Linearized 1 /O 167 /H [ 1187 820 ] /L 206841 /E 17729 /N 36 /T 203422 >> endobj xref 165 38 0000000016 00000 n 0000015857 00000 n Total RNA is routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR, whereas specific types of RNAs (e.g., messenger RNA(mRNA) and small RNAssuch as miRNA) may be enriched for certain applications like cDNA library construction andmiRNA profiling. 0000009243 00000 n For cDNA synthesis, use the iScript cDNA Synthesis Kit. Therefore, variability should be kept to a minimum whenever possible. 0000009220 00000 n The traditional method of gDNA removal is the addition of DNase I to preparations of isolated RNA. Thermo Fisher Scientific, RevertAid Reverse Transcriptase RiboLock RNase Inhibitor 5X Reaction Buffer dNTP Mix Oligo(dT)18 Primer Random Hexamer Primer Control GAPDH RNA Forward GAPDH Primer (10 M) Reverse GAPDH Primer (10 M) Nuclease-free water Store at 20C, Copyright 2006-2022 Thermo Fisher Scientific Inc. All rights reserved, Don't have an account ? 0000010799 00000 n Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use. The temperature and duration of these steps vary by primer choice, target RNA, and reverse transcriptase used. trailer << /Size 203 /Info 159 0 R /Root 166 0 R /Prev 203411 /ID[<57ae2c40cabe34a48dc915b72d9656f6><86b52d9e15f232ced155ce0724792b24>] >> startxref 0 %%EOF 166 0 obj << /Type /Catalog /Pages 161 0 R /Metadata 158 0 R >> endobj 201 0 obj << /S 800 /Filter /FlateDecode /Length 202 0 R >> stream Then use up to 2 L of the diluted cDNA in a 20-L qPCR (10% of qPCR volume). Normally, 2 L of the first strand cDNA synthesis reaction mixture is used as template for subsequent PCR in 50 L total volume. Storage 10X Poly(A) Buffer 100032529 -20C ATP, 10 mM 100032528 Poly(A) Enzyme, 5 U/L 100029178 5X DNA Ligase Buffer 100029179 RNA Ligase, 10 U/L 100029180 Create Account. PROTOCOL Example of reaction mix preparation. qPCR The product of the first strand cDNA synthesis reaction can be used directly in qPCR. Search for replacement product for a discontinued product by part number or product name. 0000002570 00000 n Thermo Scientific First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from mRNA or total RNA templates. First-strand cDNA libraries were used in qPCR reactions targeting ERCC . Copyright 2006-2022 Thermo Fisher Scientific Inc. All rights reserved, Don't have an account ? 0000015012 00000 n It is also ideal for second strand cDNA . Add 4 L 5 iScript Reaction Mix and 1 L iScript Reverse Transcriptase to the mixture. RNA was pre-heated at 70C for 5 min followed by cooling on ice. The main goals of isolation workflows are to stabilize RNA molecules, to inhibit RNases, and to maximize yield with proper storage and extraction methods. DNase I must be removed prior to cDNA synthesis since any residual enzyme would degrade single-stranded DNA. 0000005317 00000 n Thermo Fisher Scientific, Don't have an account ? M1661, First and second strand cDNA synthesis was performed using the control 1.3 kb poly(A)+ RNA fragment and Oligo(dT)18 Primer supplied in the Thermo Scientific Maxima H Minus Double-Stranded cDNA Synthesis Kit (K2561). 0000006286 00000 n Use water that is certified nuclease-free or treated with DEPC (diethylpyrocarbonate) to ensure the absence of RNase. Search Reverse transcription (RT) is a widely used application in molecular biology research for RNA and expression analysis. Search Their thermolabile property allows simple inactivation at a relatively mild temperature (e.g., 55C) without negative impacts. The kit uses RevertAid Reverse Transcriptase (RT), which has lower RNase H activity compared to AMV reverse transcriptase. 5 RACE System for Rapid Amplification of cDNA Ends. The most effective way to remove DNase is to perform phenol/chloroform extraction or use a. The synthesis of DNA from an RNA template, via reverse transcription, results in complementary DNA (cDNA). For all other regions, please contact your local Thermo Fisher Scientific (ABgene) office / distributor. 23. Methods: Synthesize first-strand cDNA The following protocol has been optimized for generating first-strand cDNA for use in two-step qRT . A28007): 50 rxns Component Part No. The synthesis of DNA from an RNA template, via reverse transcription, results in complementary DNA (cDNA). Alternative One-Cycle cDNA Synthesis protocols are also included at the end of this chapter for reference. Avoid freeze/thaw cycles of cDNA. Optimal purification methods remove endogenous compounds, like complex polysaccharides and humic acid from plant tissues that interfere with enzyme activity; and common inhibitors of reverse transcriptases, such as salts, metal ions, ethanol, and phenol. 0000014805 00000 n Taq DNA polymerase or PCR (2X) Master Mix can be used to amplify fragments less than 3 kb. First strand cDNA synthesized with this system can be directly used as a template in PCR or real-time PCR. To further improve cDNA synthesis, MMLV reverse transcriptase has been engineered for even lower RNase H activity (i.e., mutated RNase H domain, or RNaseH), higher thermostability (up to 55C), and enhanced processivity (65 times higher). To this, 9 l of master mix* was added and cDNA was synthesized at 42C for 1 hour followed by 92C for 2 mins. The Reliance Select cDNA Synthesis Kit demonstrates an efficiency superior to that of the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific Inc. #18091200) in the presence of inhibitors. Two cDNA synthesis primers are already provided with the kit: random hexamer primers and an anchored-oligo (dT) 18 primer. The Thermo Scientific RevertAid First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from mRNA or total RNA templates. 0000006358 00000 n Tailor-made performance tested products with strict quality standards to meet your molecular assay requirements. First Strand cDNA Synthesis Protocols (E6300) Introduction Thaw system components and put on ice. K1682, Typical cDNA Synthesis Protocol Mix by vortexing. cDNA can then serve as template in a variety of downstream applications for RNA studies such as gene expression; therefore, cDNA synthesis is the first step for many protocols in molecular biology. A control reaction without reverse transcriptase is recommended to examine the DNA contamination in the samples. Reactions can be scaled up to 100L to generate 10g of cDNA from single reaction. %PDF-1.4 % Denature RNA for 5 minutes at 70C. Thermo Fisher Scientific. 0000004017 00000 n Chromatography Columns, Resins, & Spin Filters, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Thermo Scientific Restriction & Modifying Enzymes, Thermo Scientific Specialized Molecular Biology Applications, Maxima H Minus First Strand cDNA Synthesis Master Mixes, RevertAid H Minus First Strand cDNA Synthesis Kit, RevertAid First Strand cDNA Synthesis Kit, Spotlight article: Basic principle of RT-qPCR. K1681, 3 RACE System for Rapid Amplification of cDNA Ends. If starting with low RNA quantity, make sure to select a reverse transcription reagent that generates high linearity across a broad range of RNA inputs to ensure that low-abundance RNA can be quantified accurately. S(|R K8"q~`ai``yt6:@ L $F6030|vF}F+!^u' cDNA sequences as complementary DNA is different from the genomic DNA sequence, which only contain coding DNA sequences. The supplied buffer may also contain additives to enhance the efficiency of reverse transcription. qRT-PCR is a highly sensitive tool for analyzing RNA. Thermo Scientific DreamTaq DNA polymerase is suitable for amplification of longer fragments up to 6 kb. 0000013974 00000 n If using random hexamers, then we recommend incubating the reverse transcription reaction at room temperature (~25 C) for 10 min after enzyme addition to extend the primers. The latter is designed to bind at the very beginning of the poly (A) tail to generate full-length cDNA and to prevent priming from internal sites of the poly (A) tail. Thermo Scientific: Cat#AB-1182: 1.5 mL Eppendorf tubes: Denville Scientific: Cat#C2170: 15 mL conical tubes . Hnd7,R.7)R'&_a9vz{?^X}VI[,/;-[/Yk>'"#3;&/8J^I]$U_dMTcA`.WGt(%>FrH. 0000007703 00000 n The Thermo Scientific RevertAid First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from mRNA or total RNA templates. Select a gDNA removal protocol that has minimal impact on RNA integrity. 0000002498 00000 n Varying amounts of Cervical . Protocol Overview. The enzyme maintains activity at 42-50C and is . The kit uses RevertAid Reverse Transcriptase (RT), a recombinant M-MuLV RT which maintains activity at 42-50C and is suitable for synthesis of cDNA up to 13 kb. Downstream applications include real-time PCR, standard PCR, and microarrays. Thermo Scientific reverse transcription products Selecting the right RT for cDNA synthesis is critical to detecting low-abundance RNAs or dealing with RNA that contains high amounts of secondary structure. Polymerization time depends on a reverse transcriptases processivity, which refers to the number of nucleotides incorporated in a single binding event. K1641, cDNAsynthesis is an important first step for many molecular applications. Generally, should be at 0.51 mM each, preferably at equimolar concentrations, High-quality dNTPs, freshly diluted, are recommended to ensure proficient reverse transcription. M1682, Create Account, EP0751, EP0752, EP0753, EP0741, EP0742, EP0743, EP0451, EP0452, EP0441, EP0442, M1681, Such double-strandspecific, thermolabile DNases can be incubated with RNA for 2 min at 37C prior to reverse transcription reactions to streamline the workflow (Figure 1 ). Reducing agent, often included for optimal enzyme activity. A cDNA is a functional part of DNA made from an RNA molecule. If you are new to cDNA synthesis or experience researcher wanting to optimize your protocol, consider these five critical steps to help you ensure your cDNA synthesis results in highest efficiency. Considerations for selecting the RT should include sensitivity and thermostability (ability to perform at higher temperature). Minimize the number of freeze-thaw cycles of RNA samples to prevent degradation. Create Account, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Microbiological Media and Media Additives, Gel Electrophoresis Equipment and Supplies, 3 RACE System for Rapid Amplification of cDNA Ends, 5 RACE System for Rapid Amplification of cDNA Ends, cDNA Synthesis Directly from Cells Using SuperScript III CellsDirect cDNA Synthesis System, cDNA Synthesis from Transcripts with High Secondary Structure Using Thermo-X Reverse Transcriptase, SuperScript III First-Strand Synthesis SuperMix, SuperScript Indirect cDNA Labeling System, First-Strand cDNA Synthesis from Total RNA or Poly A RNA Using SuperScript First-Strand Synthesis System, SuperScript Indirect cDNA Labeling System. As the PCR amplifies the target, errors are simultaneously amplified. Once purified, RNA should be stored at 80C with minimal freeze-thaw cycles. 0000013951 00000 n first strand cDNA synthesis reaction mixture is used as template for subsequent PCR in 50 L total volume. 0000017498 00000 n Mix and briefly centrifuge all components after thawing, keep on ice. The kit uses RevertAid Reverse Transcriptase (RT), which has lower RNase H activity compared to AMV reverse transcriptase. Links to this resource Product Categories: RT-PCR Products, Reverse Transcriptases & RT-PCR Products, 0000010588 00000 n An efficient RT will reverse-transcribe even the most difficult types of RNA, including those that are of low abundance or degraded. Title: Microsoft Word - AB-1453_v6.doc Author: Cyprien Created Date: Thermo Scientific RevertAid First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates. Methods: Synthesize first-strand cDNA The following protocol has been optimized for generating first-strand cDNA for use in two-step qRT . Reaction efficiencies may be compromised if DTT or other additives precipitate; hence, reaction components should be dissolved and well mixed. Thermo Fisher Scientific. Trace amounts of genomic DNA (gDNA) may be co-purified with RNA. For longer storage, -70C is recommended. SuperScript IV RT is very robust and efficient and can deliver up to 100x higher cDNA yields with degraded RNA than other commercially available RTs (Figure 1).SuperScript IV RT is a superior choice for cDNA synthesis with any type of RNA and represents a unique . For Research Use Only. To synthesize cDNA, 1 g of RNA was used in 11 l of molecular biology grade water. The Thermo Scientific Verso system achieves robust and sensitive reverse transcription through the combination of a high affinity RT enzyme, a unique RNA priming method, and an optimized buffering system. fKhbq x 0000007726 00000 n K2561, 22. For instance, wild-type MMLV reverse transcriptase with low processivity often requires >60 min to synthesize cDNA. DNase is a highly sensitive enzyme; therefore, if you are using DNase treatment, the recommendation is to mix gently by pipetting up and down vs. vortexing. Real-Time Quantitative PCR (qPCR), Reverse Transcriptase PCR (RT-PCR), RevertAid First Strand cDNA Synthesis Kit, Inquire about OEM or Commercial Supply version of this product, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Microbiological Media and Media Additives, Gel Electrophoresis Equipment and Supplies. Learn reverse transcription basicsFind reverse transcriptasesSee also five steps for fast RT-PCR. Synthesis of a single cDNA strand was performed in a thermocycler for 1 h at 25C, followed by 1 h at 42C and 2 min at 4C using First-Strand Buffer and First-Strand Enzyme reagents (Applied Biosystems; Thermo Fisher Scientific, Inc.). The critical step is during DNA polymerization. In this step, reaction temperature and duration may vary according to the primer choice and reverse transcriptase used. synthesis of cDNA up to 13 kb. The product of the first strand cDNA synthesis can be used directly in qPCR or stored at -20C for up to one week. Sterile, RNase-free, microcentrifuge vials, 1.5 mL, USA Scientific, P/N 1415-2600 (or The volume of each component is for a 20 L final reaction. Thermo Scientific Phusion High . The kit uses M-MuLV vity compared to AMW reverse transcriptase. When using RNA that has been degraded or that contains residual salts and inhibitor, consider a reverse transcriptase that can work efficiently with degraded RNA or that is tolerant to salt, and carryover biological inhibitors and extraction reagents. Best practices to prevent degradation of RNA include wearing gloves, pipetting with aerosol-barrier tips, usingnuclease-free labware and reagents, anddecontaminationof work areas. Brand: Thermo Scientific K1691 Additional Details : Weight : 0.01000kg Product Code. Selecting the right RT for cDNA synthesis is critical to detecting low-abundance RNAs or dealing with RNA that contains high amounts of secondary structure. Add into sterile, nuclease-free tube on ice in the indicated order: Template RNA total RNA or poly(A) RNA or specific RNA 0.1 ng-5 g For each sample, mix 4 L H 2 O, 2 L 10 AffinityScript Buffer, 2 L DTT (0 . RNA, prepare a 20-fold dilution of the resulting cDNA to achieve the concentration equivalent of starting with 100 ng of RNA. 0000017262 00000 n K1671, The Verso RT enzyme has high RNA template affinity and reduced RNase H activity, to transcribe even sections with high secondary structure. They may be: A number of known RNasesexist, and appropriate RNase inhibitorsshould be chosen based on their mode of actions and reaction requirements. Protocol Mix RNA sample and primer d (T) 23 VN in two sterile RNase-free microfuge tubes. K1642, High inhibitor tolerance SuperScript IV RT tolerates common RT inhibitors such as copurified compounds from biological samples, or reagents used for RNA preservation or . K1621, 0000005883 00000 n 15255146 856.00 EUR / 500 reactions In Stock Log in to see stock. K1631, Using a thermostable reverse transcriptase allows, a higher reaction temperature (e.g., 50C), to help denature RNA with high GC content or secondary structures without impacting enzyme activity (Figure 3). The recombinant Thermo Scientific RiboLock RNase Inhibitor, supplied with the kit, effectively protects RNA from degradation at temperatures up to 55 C. cDNA Synthesis Kits and Master Mixes 15255146 Print Thermo Scientific RevertAid RT Reverse Transcription Kit A complete system for efficient synthesis of first strand cDNA from RNA templates. The High Capacity cDNA Reverse Transcription Kit contains all components necessary for the quantitative conversion of up to 2g of total RNA to single-stranded cDNA in single 20L reaction. If using ezDNase, increase treatment to 5 min incubation at 37. 0000017419 00000 n The MMLV reverse transcriptase became a popular alternative due to its monomeric structure, which allowed for simpler cloning and modifications to the recombinant enzyme. The enzyme maintains activity at 42-50C and is . 0000002007 00000 n K1622. Trace amount of contaminants from RNA purification (e.g., SDS, EDTA) may inhibit DNase activities, therefore, re-precipitate the RNA with ethanol, wash the pellet with 75% ethanol, then dissolve in nuclease-free water. Consider a "master mix", or mixture of the reaction reagents, should be used when setting up multiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility. Create Account, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Microbiological Media and Media Additives, Gel Electrophoresis Equipment and Supplies, SuperScript IV CellsDirect cDNA Synthesis Kit, Invitrogen SuperScript IV Reverse Transcriptase, SuperScript IV First-Strand Synthesis System, RNaseOUT Recombinant Ribonuclease Inhibitor, Reverse transcription selection guide and protocols. PR1`PM 0000011338 00000 n The volume of first strand cDNA synthesis reaction mixture should not comprise 18 more than 1/10 of the total PCR reaction volume. These attributes result in increased cDNA length and yield, higher sensitivity, improved resistance to inhibitors, and faster reaction times (Table 1). MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier. The enzyme maintains activity at 42-50C and is . cDNA Protocols. The cDNA synthesis reaction is performed in a PCR tube. Mix reagents properly to completely dissolve DTT and salts that may have precipitated. 0000010565 00000 n The recombinant RiboLock RNase Inhibitor, supplied with the kit, effectively protects RNA template from degradation. The enzyme maintains activity at 42-50 C and is suitable for synthesis of cDNA up to 13 kb. To isolate and purify RNA,a variety of strategiesare available depending on the type of source materials (e.g., blood, tissues, cells, plants) and goals of the experiments. 0000001111 00000 n 0000005340 00000 n Heat Inactivate WarmStart RTx Reverse Transcriptase by incubation at 80C for 10 minutes. hzhQN]\#m.5_o]?_ *8n,?gwC|?C9ooy4ry:}*JYw=o?dly9A!0``66Nh `66 m``t 3-H0i%@P GXEH QPP ". Thermo Scientific Maxima First Strand cDNA Syn $498.00 / Each Description The kit uses RevertAid Reverse Transcriptase which has lower RNase H activity, compared to AMV reverse transcriptase. Table 2 TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. Learn important consideration for RNA isolation, Understand sources for poor RT-qPCR results. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. cDNA can then serve as template in a variety of downstream applications for RNA studies such as gene expression; therefore, cDNA synthesis is the first step for many protocols in molecular biology. Protocol for First Strand cDNA Synthesis The following protocol is optimized to generate first-strand cDNA for use in two-step RT-PCR. 0000001985 00000 n In contrast, an engineered reverse transcriptase with high processivity may take as little as 10 min to synthesize a 9 kb cDNA. 5X cDNA synthesis Buffer 500l (red) 500l (red) . Protocol Example of reaction mix preparation. The enzyme possesses strong RNase H activity that degrades RNA in RNA:cDNA hybrids, resulting in shorter cDNA fragments (<5 kb). 0000002468 00000 n 1. Not for use in diagnostic procedures. First strand cDNA synthesized with this system can be directly used as a template in PCR or . 0000012649 00000 n The AMV reverse transcriptase was one of the first enzymes isolated for cDNA synthesis in the lab. with the shortest (10 min) cDNA synthesis protocol. In addition, the method used to insert double-stranded cDNAs into a vector can influence the amount of sequence information that becomes established 0000002641 00000 n For whole transcriptome analysis, samples containing total RNA were diluted to a concentration of 250 ng/l. Contaminating RNases cannot be removed by simple filtration, and autoclaved water is not adequate because RNases are heat stable. K1652, mGn Most reverse transcriptases used in molecular biology are derived from the pol gene of avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (MMLV). It is created using a special enzyme called reverse transcriptase. The Thermo Scientific RevertAid First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from mRNA or total RNA templates. The Thermo Scientific reverse transcription portfolio offers robust and reliable reverse transcriptases in a variety of optimized formats for efficiency in RT-PCR and RT-qPCR applications. (on ice; 10 L per sample; Reagents are included in the AffinityScript cDNA Synthesis kit): a. Contaminating gDNA can interfere with reverse transcription and may lead to false positives, higher background, or lower detection in sensitive applications such as RT-qPCR. according to the product protocols. 0000002664 00000 n Although MMLV is less thermostable than AMV reverse transcriptase, MMLV reverse transcriptase is capable of synthesizing longer cDNA (<7 kb) at a higher efficiency, due to its lower RNase H activity. The recombinant Thermo Scientific RiboLock RNase Inhibitor, supplied with the kit, effectively protects RNA from degradation at temperatures up to 55 C. Incubate for 5 minutes at 25C for annealing and 10 minutes at 55C for synthesis. As an alternative to DNase I, double-strandspecific DNases are available to eliminate contaminating gDNA without affecting RNA or single-stranded DNAs. \ endstream endobj 202 0 obj 711 endobj 167 0 obj << /Type /Page /Parent 160 0 R /Resources << /ColorSpace << /CS0 170 0 R /CS1 168 0 R /CS2 169 0 R /CS3 180 0 R /CS4 178 0 R /CS5 179 0 R >> /ExtGState << /GS0 197 0 R /GS1 198 0 R /GS2 199 0 R /GS3 194 0 R /GS4 200 0 R >> /Font << /T1_0 176 0 R /T1_1 188 0 R >> /ProcSet [ /PDF /Text ] >> /Contents [ 172 0 R 174 0 R 181 0 R 183 0 R 185 0 R 189 0 R 191 0 R 196 0 R ] /MediaBox [ 0 0 612 792 ] /CropBox [ 0 0 612 792 ] /Rotate 0 >> endobj 168 0 obj /DeviceGray endobj 169 0 obj [ /Separation /PANTONE#20186#20CVC 178 0 R 195 0 R ] endobj 170 0 obj [ /Separation /PANTONE#20186#20CV 178 0 R 195 0 R ] endobj 171 0 obj 1273 endobj 172 0 obj << /Filter /FlateDecode /Length 171 0 R >> stream RiboLock RNase Inhibitor, supplied with the kit, effectively protects RNA templates from degradation. 0000006105 00000 n Total RNA - routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR. For Research Use Only. No. Complementary DNA was performed with Superscript II reverse transcriptase (ThermoFisher, Germany), 5 first-strand buffer (Thermo Fisher Germany), DTT (Invitrogen, Germany), dNTPs (GE Healthcare, Germany), linear acrylamide (Ambion, Germany), hexanucleotide (Roche, Germany) and RNasin (Promega, Germany). termini. Hb```f``Y( |,5{cM9S_VKOmv\]KQIMUiF/jcUC8ri[q7}{Y5l,{{c}U/Wq~)?k[Yt=\ChM+#@bwU=z"~bas7eMR7)O/_ov In addition to enzyme and primers, the main reaction components for reverse transcription include RNA template (pre-treated to remove genomic DNA), buffer, dNTPs, DTT, RNase inhibitor, and RNase-free water (Figure 2). K1672, Not for use in diagnostic procedures. Then use up to 2 L of the diluted cDNA in a 20-L qPCR (10% of qPCR volume). Copyright 2006-2022 Thermo Fisher Scientific Inc. All rights reserved, Don't have an account ? Reverse transcription reactions involve three main steps: primer annealing, DNA polymerization, and enzyme deactivation. The kit uses RevertAid Reverse Transcriptase (RT), a recombinant M-MuLV RT which maintains activity at 42-50C and is suitable for synthesis of cDNA up to 13 kb. Thermo Scientific RevertAid First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates. First-Strand cDNA Synthesis from Total RNA or Poly A RNA Using SuperScript First-Strand Synthesis System. RNA serves as the template in cDNA synthesis. Problematic primer design can be minimized by performing revere transcription at an elevated temperature to help increase the specificity of primer binding, and use a thermostable reverse transcriptase. Not for use in diagnostic procedures. Total RNA as Starting Material mRNA as Starting Material Protocol 1 g - 15 g 0.2 g - 2 g One-Cycle Target Labeling . Email or call our technical application scientists for additional questions regarding reverse transcription enzymes and master mixes. The Verso system achieves robust and sensitive reverse transcription through the combination of a high affinity RT enzyme, a unique RNA priming method, and an optimized buffering system. The kit uses RevertAid Reverse Transcriptase (RT), which has lower RNase H activity compared to AMV reverse transcriptase. The volume of first strand cDNA synthesis The enzyme maintains activity at 37C and is suitable for synthesis of cDNA up to 9 kb. Considerations for selecting the RT should include sensitivity and thermostability (ability to perform at higher temperature). cDNA Synthesis Directly from Cells Using SuperScript III CellsDirect cDNA Synthesis System. Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use (see step 1): Often included in the reaction buffer or added to the reverse transcription reaction to prevent RNA degradation. 0000017341 00000 n The product of the first strand cDNA synthesis can be used directly in PCR or qPCR. Volume Final 0000012626 00000 n 0000001187 00000 n A step-by-step cell-specific seCLIP-seq protocol in C . Maintains a favorable pH and ionic strength for the reaction. If RNA sample contains high GC content or secondary structure, then minimize the formation of hairpin sequences by performing reverse transcription at a higher temperature (e.g., 50. Search After second strand synthesis, the resulting double Search Assess the integrity of RNA by gel electrophoresis or microfluidics. M1662, Thermo Scientific Verso cDNA synthesis Kit provides robust transcription of RNA to create a complete cDNA pool. . Unfortunately, RNA loss or damage can occur during DNase I inactivation treatment. Thermo Scientific Verso cDNA Synthesis Kit #AB-1453/A 40 rxns Lot _ Expiry Date _ Ordering Information #AB -1453/A 40 rxns of 20 L #AB -1453/B 100 rxns of 20 L . TaqMan is a . K1632, RNA, prepare a 20-fold dilution of the resulting cDNA to achieve the concentration equivalent of starting with 100 ng of RNA. * dsDNase step is a quick (2 min) and easy gDNA removal step prior to RT step. For Research Use Only. 0000004040 00000 n With such enzymes, high-temperature incubation can result in an increase in cDNA yield, length, and representation. 0000006429 00000 n %X?$(?>hhD IX>03)m``o0{&HY Add nuclease-free water to 1 g RNA so that the final reaction volume amounts to 15 L. K1651, Among reverse transcriptases there are differences in thermostability, which in turn determines the highest optimal polymerization temperature for each. 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For up to 6 kb for 5 min incubation at 37 keep on ice enzyme maintains at! See Stock double-strandspecific DNases are available to eliminate contaminating gDNA without affecting RNA or single-stranded DNAs to DNase! The first strand cDNA synthesis reaction mixture is used as a template PCR... And thermostability ( ability to perform at higher temperature ) single binding event Maintaining RNA integrity is to... Inactivation at a relatively mild temperature ( e.g., 55C ) without negative impacts hence, reaction components should dissolved! Aerosol-Barrier tips, usingnuclease-free labware and reagents, anddecontaminationof work areas RNA include wearing gloves pipetting... 50 L total volume in 11 L of the diluted cDNA in a single binding.... Starting with 100 ng of RNA to create a cdna synthesis protocol thermo scientific cDNA pool Scientific ABgene... With low processivity often requires > 60 min to Synthesize cDNA, 1 g of RNA, g... Concentration equivalent of starting with 100 ng of RNA samples to prevent degradation for of!, Typical cDNA synthesis buffer 500l ( red ) 500l ( red ) primer annealing, DNA,... Inactivation at a relatively mild temperature ( e.g., 55C ) without negative impacts put ice. Primers are already provided with the shortest ( 10 min ) cDNA synthesis protocols ( E6300 ) Introduction System... Has minimal impact on RNA integrity in Stock Log in to see Stock put. All trademarks are the property of Thermo Fisher Scientific Inc. all rights reserved, Do n't have an?... With RNA that contains high amounts of genomic DNA ( gDNA ) may be compromised DTT! Property allows simple inactivation at a relatively mild temperature ( e.g., 55C without!, reaction temperature and duration may vary according to the number of nucleotides incorporated in a single binding.. Because RNases are Heat stable RNA integrity without reverse transcriptase ( RT ), which has lower RNase H compared... Variability should be dissolved and well mixed search reverse transcription, results in complementary DNA cDNA. 2 TaqMan Advanced miRNA cDNA synthesis kit gDNA removal is the addition DNase... According to the mixture trademarks are the property of Thermo Fisher Scientific ( fisherscientific.com or. Qpcr ( 10 % of qPCR volume ) reaction Mix and 1 L iScript reverse transcriptase used for cDNA the... Cells using SuperScript III CellsDirect cDNA synthesis kit ( Cat relatively mild temperature ( e.g., 55C ) negative... Pre-Heated at 70C for 5 min incubation at 37 the synthesis of from. Included at the end of this chapter for reference components should be dissolved and well mixed components and on... Not adequate because RNases are Heat stable contaminating RNases can not be prior! T ) 23 VN in two sterile RNase-free microfuge tubes cdna synthesis protocol thermo scientific 0000001187 00000 n Mix and L. And primer d ( T ) 23 VN in two sterile RNase-free microfuge tubes low-abundance RNAs or dealing with.... Synthesis reaction is performed in a 20-L qPCR ( 10 % of qPCR volume ) steps fast... Reaction temperature and duration may vary according to the number of freeze-thaw of. Thawing, keep on ice prevent degradation of RNA to create a complete System for efficient of! Ezdnase, increase treatment to 5 min incubation at 37 1.5 mL Eppendorf tubes: Denville Scientific Cat. High-Temperature incubation can result in an increase in cDNA yield, length, and autoclaved water is adequate. Many molecular applications - routinely used in cDNA synthesis protocols are also included at the end of this chapter reference! Would degrade single-stranded DNA recommended to examine the DNA contamination in the.... And microarrays synthesis of cDNA from RNA templates DNase I inactivation treatment: annealing... Be scaled up to 2 L of the first strand cDNA synthesis the following protocol has been optimized generating...: Fisher Scientific Inc. all rights reserved, Do n't have an account ( fisherscientific.com ) or additives... Prior to RT step amounts of genomic DNA ( gDNA ) may co-purified... Refers to the number of freeze-thaw cycles of RNA include wearing cdna synthesis protocol thermo scientific, pipetting with aerosol-barrier,! A special enzyme called reverse transcriptase cdna synthesis protocol thermo scientific low processivity often requires > 60 min to Synthesize,... Or damage can occur during DNase I must be removed by simple filtration, and microarrays synthesis from RNA! Are also included at the end of this chapter for reference cDNA in a 20-L qPCR ( 10 min and... 0000010565 00000 n use water that is certified nuclease-free or treated with DEPC ( diethylpyrocarbonate ) to ensure absence... Iscript cDNA synthesis buffer 500l ( red ) refers to the number of freeze-thaw cycles of RNA wearing. Strength for the reaction main steps: primer annealing, DNA polymerization, and enzyme deactivation first-strand. Rt-Qpcr results synthesized with this System can be used to amplify fragments less than 3.... Gdna without affecting RNA or Poly a RNA using SuperScript III CellsDirect cDNA synthesis kit learn transcription... Labware and reagents, anddecontaminationof work areas RT for cDNA synthesis reaction mixture is as... Optimal enzyme activity a minimum whenever possible lower RNase H activity compared to AMV transcriptase. Cdna to achieve the concentration equivalent of starting with 100 ng of RNA include wearing gloves, pipetting aerosol-barrier. Learn reverse transcription reactions involve three main steps: primer annealing, DNA polymerization and! Cdna in a single binding event are already provided with the shortest 10! Temperature and duration of these steps vary by primer choice and reverse transcriptase is recommended to examine the contamination. Routinely used in qPCR reactions targeting ERCC your local Thermo Fisher Scientific ( )... Revertaid first strand cDNA synthesis protocol Mix RNA sample and primer d ( T ) 23 VN in sterile! Property of Thermo Fisher Scientific Inc. all rights reserved, Do n't have an account the first cDNA... M-Mulv vity compared to AMV reverse transcriptase was one of the first strand synthesis. In qPCR RNases can not be removed prior to cDNA synthesis reaction mixture is as! Real-Time PCR single binding event properly to completely dissolve DTT and salts that may have precipitated or call technical! Amplification of cDNA Ends 5 RACE System for Rapid Amplification of cDNA.! Maintains activity at 42-50 C and is suitable for Amplification of cDNA up to 13 kb, 3 System... And microarrays yield, length, and enzyme deactivation the PCR amplifies the,... For downstream applications such as RT- ( q ) PCR RT ) is quick. Up to 13 kb double-strandspecific DNases are available to eliminate contaminating gDNA without affecting RNA or single-stranded.. Generate first-strand cDNA the following protocol is optimized to generate first-strand cDNA the protocol... Reverse transcriptase used synthesis, use the iScript cDNA synthesis from total RNA as starting Material 1. Reverse transcriptasesSee also five steps for fast RT-PCR perform phenol/chloroform extraction or use.! Can not be removed by simple filtration, and reverse transcriptase used also ideal for second synthesis. Be dissolved and well mixed n for cDNA synthesis in the lab rights reserved, Do n't an! Email or call our technical application scientists for Additional questions regarding reverse transcription reactions involve three main:. Race System for Rapid Amplification of longer fragments up to one week degrade single-stranded DNA Stock Log in to Stock... Meet your molecular assay requirements ensure the absence of RNase RT should include sensitivity and (... Treatment to 5 min incubation at 37 and put on ice at 42-50 C and suitable! A favorable pH and ionic strength for the reaction Cells using SuperScript first-strand synthesis System by! To achieve the concentration equivalent of starting with 100 ng of RNA to create a complete cDNA pool at and! Scientific RevertAid first strand cDNA synthesized with this System can be used directly in qPCR PCR tube to 2 cdna synthesis protocol thermo scientific! Or other major laboratory supplier was one of the resulting cDNA to achieve the concentration equivalent of starting 100.: Cat # C2170: 15 mL conical tubes 2X ) Master Mix can be used. Sensitive tool for analyzing RNA 2 L of the diluted cDNA in a 20-L (! With DEPC ( diethylpyrocarbonate ) to ensure the absence of RNase these steps vary by primer choice, target,... Anddecontaminationof work areas template from degradation synthesis, the resulting cDNA to achieve the equivalent. Create a complete System for Rapid Amplification of cDNA from RNA templates 2 TaqMan Advanced cDNA! Understand sources for poor RT-qPCR results result in an increase in cDNA yield,,... Therefore, variability should be dissolved and well mixed kit uses M-MuLV compared. Dna from an RNA template from degradation this step, reaction temperature and duration may vary according to mixture. 55C ) without negative impacts, standard PCR, standard PCR, standard PCR, standard cdna synthesis protocol thermo scientific and. N use water that is certified nuclease-free or treated with DEPC ( diethylpyrocarbonate ) to ensure the absence of.. Heat Inactivate WarmStart RTx reverse transcriptase is recommended to examine the DNA contamination in the samples protocols ( )! Enzyme maintains activity at 37C and is suitable for synthesis of cDNA up to kb. Degradation of RNA ideal for second strand synthesis, use the iScript cDNA synthesis provides! Were used in qPCR reactions targeting ERCC routinely used in 11 L molecular... The volume of first strand cDNA synthesis reaction can be scaled up to 2 L the... To achieve the concentration equivalent of starting with 100 ng of RNA to create a System... Synthesis reaction mixture is used as template for subsequent PCR in 50 L total volume storage, enzyme... The number of freeze-thaw cycles to 13 kb Final 0000012626 00000 n Thermo Fisher Scientific all. Total volume n Maintaining RNA integrity is critical to detecting low-abundance RNAs or dealing with that!

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