HiFiScript cDNA Synthesis Kit

Cat. No.: H665509

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Vue d’ensemble

H665509	Component	100 T	Storage
H665509A	HiFiScript, 200 U/μL	100 μL	-20℃. Avoid freeze/thaw cycle.
H665509B	5×RT Buffer	500 μL	-20℃. Avoid freeze/thaw cycle.
H665509C	Primer Mix	240 μL	-20℃. Avoid freeze/thaw cycle.
H665509D	dNTP Mix, 2.5 mM Each	500 μL	-20℃. Avoid freeze/thaw cycle.
H665509E	DTT, 0.1 M	240 μL	-20℃. Avoid freeze/thaw cycle.
H665509F	RNase-Free Water	1 mL	-20℃. Avoid freeze/thaw cycle.

Product Introduction

This product is a cDNA first-strand synthesis kit specially formulated for the first step of two-step RT-PCR. The HiFiScript Reverse Transcriptase used in this kit is a new high-efficiency reverse transcriptase derived from M-MLV, with novel mutation sites that dramatically improve the transcriptional activity of the enzyme, and the efficiency and yield of cDNA first-strand synthesis is higher, and the first strand of cDNA can be synthesized using pg-level total RNA or mRNA. The point mutation eliminates RNase H activity and improves the elongation performance of the enzyme, which effectively improves the affinity of the reverse transcriptase to the RNA template, and allows for the production of cDNAs up to 12 kb. The carefully optimized RT Buffer allows for a wider range of applications of the reverse transcriptase, and provides high compatibility for subsequent PCR and quantitative PCR experiments. The kit comes with all reverse transcription reagents and is easy to use. It is suitable for first-strand cDNA synthesis and subsequent RT-PCR, RT-qPCR, and full-length cDNA library construction.

Product Features

1. Highly efficient reverse transcription efficiency: novel mutation sites dramatically increase enzyme activity for higher yields of cDNA.

2. Good elongation: point mutation eliminates RNase H activity and improves the affinity of reverse transcriptase for RNA templates, resulting in cDNAs up to 12 kb long.

3. High sensitivity: cDNA first strand can be synthesized using pg-level total RNA or mRNA templates.

4. Easy to use: The Reverse Transcription Kit comes with all the reverse transcription reagents, so you can easily complete the reverse transcription by simply preparing the template.

matters needing attention

1. RNase contamination should be avoided during operation to prevent RNA degradation or cross-contamination in the experiment. It is recommended that RNA operations be performed in a dedicated area, specialized instruments and consumables be used, and that operators wear masks and disposable gloves and change gloves frequently.

2. Disposable plasticware should be used as much as possible. If glassware is used, it should be treated with 0.1% DEPC (diethylpyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved at 120℃ for 30 minutes before use, or the glassware should be dry heat sterilized at 180℃ for 60 minutes before use. Sterile water used in the experiment should be treated with 0.1% DEPC and then autoclaved.

3. All reagents in this kit should be mixed gently before use by turning up and down to avoid foaming as much as possible, and after brief centrifugation. The enzymes involved should be put back to -20℃ as soon as possible after use to avoid repeated freezing and thawing.

If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).

Usage

Note: 1ng-5μg of total RNA can establish a 20μl reaction system, if the amount of total RNA is greater than 5μg, please expand the reaction system proportionally.

Reverse transcription procedure:

1. Dissolve RNA template, Primer Mix, dNTP Mix, DTT, RT Buffer, HiFiScript and RNase-Free Water and set aside on ice.

2. Prepare the reaction system according to the following table in a total volume of 20 μl.

Reagents	20 μl Reaction system	Final concentration
dNTP Mix，2.5 mM Each	4 μl	500 μM Each
Primer Mix	2 μl
RNA Template	X μl
5×RT Buffer	4 μl	1×
DTT，0.1 M	2 μl	10 mM
HiFiScript，200 U/μl	1 μl
RNase-Free Water	up to 20 μl

Note: 1) If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).

2) Primer Mix was prepared from Oligo (dT) and Random Primer.

3. Vortex and shake to mix and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.

4. cDNA synthesis reaction conditions:

1) If fluorescent quantitative PCR assay is performed downstream, incubate at 42°C for 15 minutes and 85°C for 5 minutes.

2) If downstream for normal PCR assay, incubate at 42°C for 30-50 minutes and 85°C for 5 minutes.

Note: For templates with complex secondary structure or high GC content, the reverse transcription temperature can be increased to 50°C to enhance reverse transcription efficiency.

5. At the end of the reaction, centrifuge briefly and place on ice to cool.

6. The reverse transcription product can be directly used in PCR reaction and fluorescence quantitative PCR reaction, or placed in -20℃ for long-term storage.

The following steps are recommended if reverse transcription efficiency is low, or if the RNA template secondary structure is complex and GC content is high:

1. Dissolve RNA template, Primer Mix, dNTP Mix, DTT, RT Buffer, HiFiScript and RNase-Free Water and set aside on ice.

2. Prepare the reaction system according to the following table in a total volume of 13 μl

Reagents	20 μl Reaction system	Final concentration
dNTP Mix，2.5 mM Each	4 μl	500 μM Each
Primer Mix	2 μl
RNA Template	X μl	50 pg-5μg
RNase-Free Water	up to 13 μl

3. Incubate at 70°C for 10 minutes and rapidly ice bath for 2 minutes.

4. Centrifuge briefly so that the solution on the walls of the tube collects at the bottom.

5. Continue to add the following reagents to the above reaction solution:

Reagents	20 μl Reaction system	Final concentration
5×RT Buffer	4 μl	1×
DTT，0.1 M	2 μl	10 mM
HiFiScript（200 U/μl）	1 μl

Note: 1) If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).

2) Primer Mix was prepared from Oligo (dT) and Random primer.

6. Perform cDNA first-strand synthesis:

1) If fluorescent quantitative PCR assay is performed downstream, incubate at 50°C for 15 minutes and 85°C for 5 minutes.

2) If downstream for normal PCR assay, incubate at 50°C for 30-50 minutes and 85°C for 5 minutes.

7. At the end of the reaction, centrifuge briefly and place on ice to cool.

8. The reverse transcription product can be used directly in PCR reaction and fluorescence quantitative PCR reaction, the amount added should be less than 1/10 of the PCR reaction system, or placed in -20℃ for long-term storage.

Spécifications

Conditions de stockage de stockage

Store at -20°C,Avoid repeated freezing and thawing

Expédié en

Ice chest + Ice pads

Ce produit nécessite l'expédition en chaîne froide. Les services terrestres et autres services économiques ne sont pas disponibles.

Product Line

Produits biologiques

Application Protocols

No manufacturer‑validated protocols are provided in the Product Data for this item. The following is an illustrative, general workflow for first‑strand cDNA synthesis (literature/general guidance; not item‑specific):

Example workflow (20 µL total)

1. On ice, combine:
   • RNA (up to 1 µg total RNA) + primer (e.g., 50–100 ng oligo(dT) or 100 ng random hexamers) + dNTPs (final 0.5 mM each). Bring to 10 µL with RNase‑free water. Heat 65°C 5 min, quick‑chill on ice.
2. Add 10 µL of master mix containing:
   • 1× RT buffer (as supplied by kit; composition not specified here)
   • Mg2+ per buffer formulation
   • RNase inhibitor (optional, 20–40 U)
   • Reverse transcriptase (per kit recommendation; units/concentration not specified)
3. Incubate:
   • 42–55°C for 10–60 min (enzyme‑dependent)
   • Optional inactivation 70–85°C for 5–10 min (enzyme‑dependent)
4. Downstream use:
   • Use 1–2 µL cDNA per 20 µL qPCR; or proceed to second‑strand synthesis/library prep as required.

Controls

• No‑RT control (omit enzyme) and no‑template control (water) to assess gDNA contamination and reagent sterility.

Always follow the official insert for this kit when available; the above is non‑binding guidance.

Biological Roles

This product facilitates the enzymatic conversion of RNA to complementary DNA (cDNA) using a reverse transcriptase. Biological roles and context are provided as general background (not item‑specific composition or claims).

• Reverse transcriptase (RT): An RNA‑dependent DNA polymerase that synthesizes a DNA strand complementary to an RNA template. Many engineered RTs are derived from retroviral enzymes but modified for enhanced thermostability, processivity, and fidelity.
• cDNA in research: cDNA is a DNA representation of expressed RNA, enabling stable amplification, cloning, and sequencing without RNA’s lability.
• Priming strategies: Oligo(dT) targets polyadenylated mRNA; random primers enable more uniform transcript coverage; gene‑specific primers enrich for targets of interest.
• Reaction milieu: Magnesium ions, dNTPs, and optimized buffers support polymerase catalysis; RNase inhibitors protect RNA from degradation.
• Downstream biology: cDNA serves as a template for PCR/qPCR, enabling quantification of transcript abundance and analysis of splicing and isoforms.

Note: No medical or clinical utility is claimed. All uses are for research in molecular biology workflows.

Buffer Applications

Buffer selection within a cDNA synthesis workflow affects priming efficiency, enzyme fidelity, and RNA integrity. Exact buffer composition for this item is not specified; consult the kit insert/CoA.

General buffer considerations (literature/general)

• pH and ionic strength: Tris‑HCl buffers near pH 7.5–8.5 with appropriate salt (e.g., K+ or Na+) often support reverse transcription while preserving primer annealing.
• Divalent cations: Mg2+ is typically essential; concentration tuning can influence yield and length. Mn2+ can increase misincorporation and is generally avoided for high‑fidelity synthesis.
• Reducing environment: DTT (1–10 mM) is sometimes included to stabilize enzymes; compatibility depends on the specific RT and downstream assays.
• RNase inhibitor: Beneficial when working with low‑abundance or precious RNA samples.
• Additives: Betaine, DMSO, or mild detergents can aid difficult templates; use only if validated for the specific kit (not specified here).

Practical tips

• Prepare master mixes to minimize pipetting error and exposure time at room temperature.
• Use nuclease‑free tubes and water; pre‑chill racks and reagents.
• Include appropriate negative controls to assess buffer or water contamination.

Green Alternatives

While enzymatic cDNA synthesis kits are aqueous and generally avoid hazardous organic solvents during reaction setup, sustainability considerations still apply to sample prep and workflow design.

Greener practice options (general guidance)

• Reduce cold‑chain impact: If workflow allows, consolidate orders and minimize dry‑ice shipments. Some formulations from various suppliers are room‑temperature stable; stability for this item is not specified.
• Plastic reduction: Use multi‑well formats, low‑waste tip strategies, and recyclable secondary packaging where permitted by contamination control.
• Solvent‑free cleanup: Prefer column/bead‑based cleanup over phenol‑chloroform. If organic extraction is unavoidable, limit volumes and ensure proper waste segregation.
• Energy efficiency: Plan batch processing to reduce freezer door‑open time and instrument idle time.

Comparison (general, not item‑specific)

• Phenol/Chloroform RNA purification vs. column/beads:
  • Hazard profile: High vs. low
  • Aqueous waste: Low vs. moderate
  • Performance: Both can yield high‑quality RNA; columns/beads typically safer and faster.

Compliance and documentation

• Track lot numbers and QC data electronically to reduce paper use.
• Review institutional green lab guidelines for cold storage management and plastic recycling.

Pharmaceutical Uses

This product is a research‑use‑only cDNA synthesis kit and is not intended for clinical diagnostics, therapeutic, or GMP manufacturing applications.

General formulation/bioprocess context (not item‑specific)

• Developmental assays: Reverse transcription is used in R&D gene expression studies supporting target discovery and characterization.
• QC and analytics: cDNA generation can support transcript verification during cell line development or process characterization in non‑GMP settings.
• Technology transfer: For translational or regulated workflows, validated, GMP‑compliant kits and assays with documented traceability and lot release criteria are required; this item’s compliance status is not specified.

Notes

• Research Use Only (per Product Data). Users are responsible for determining suitability in their laboratory environment.
• For regulated applications, consult your quality unit and select reagents with appropriate certifications and change‑control documentation.

Physical Properties

As a multi‑component molecular biology kit, conventional physical constants are not defined at the kit level.

Item‑specific properties

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• pH, density, refractive index, melting/boiling points: Not specified for this item; refer to CoA/Spec Sheet.

General expectations for similar kits (literature/general guidance; not item‑specific)

• Enzyme solutions are commonly supplied in aqueous buffers with cryo‑/stabilizers and may remain fluid at subzero temperatures when glycerol is present; however, do not infer composition for this item without the CoA.
• Buffers for cDNA synthesis are typically aqueous, near‑neutral to slightly alkaline (pH ~7.5–8.5), RNase‑free.
• dNTP stocks are usually provided as aqueous solutions (e.g., 10 mM each) or premixes; concentration for this item is not specified.

Handling and stability considerations (general)

• Keep all components cold during setup (on ice) to preserve enzyme activity and RNA integrity.
• Avoid foam and vigorous vortexing of enzyme vials to prevent denaturation.
• Thaw on ice and mix gently by pipetting or brief, low‑speed vortexing; quick‑spin to collect condensate.

For definitive values (concentrations, pH, compositions, and any stabilizers), consult the product insert, CoA, and SDS.

Quality and Grades

Item‑specific grade/purity is not provided in the Product Data.

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.

General quality expectations for cDNA synthesis kits (literature/industry practice; not item‑specific)

• Nuclease status: Kits are typically qualified RNase‑free and DNase‑free to protect RNA integrity and ensure accurate first‑strand synthesis.
• Functional QC: Lots are commonly tested in reverse‑transcription reactions across a dynamic RNA input range and assessed by endpoint PCR or qPCR for sensitivity, linearity, and reproducibility.
• Inhibitor tolerance: High‑performance (“HiFi”) RT enzymes are often engineered for reduced RNase H activity, enhanced thermostability, and improved processivity/fidelity; actual properties for this item should be confirmed on the CoA.
• Low genomic DNA background: Some workflows include a genomic DNA removal step (e.g., DNase treatment); inclusion in this kit is not specified.

Implications for users

• For quantitative work (RT‑qPCR, RNA‑seq), review the lot‑specific CoA for RNase testing, recommended input amounts, and primer compatibility.
• If low UV background or specific UV cutoffs are relevant to downstream analytics, consult the CoA/SDS; these parameters are not specified for this item.

Reaction and Applications

This kit is intended for first‑strand cDNA synthesis from RNA templates for research use (per Product Data). While item‑specific compositions and performance claims are not provided, the following summarizes typical applications and best practices for cDNA synthesis (literature/general guidance).

Core applications

• RT‑qPCR: Convert mRNA or total RNA to cDNA for gene expression quantification.
• RNA profiling and discovery: Library preparation steps for RNA‑seq often begin with cDNA synthesis.
• Cloning and gene construction: Generate cDNA for ORF cloning, RACE, and isoform analysis.
• Low‑input or degraded RNA workflows: Thermostable, high‑processivity RTs can improve yield from challenging templates (verify properties for this item on the CoA).

Practical tips

• Primer strategy: Choose oligo(dT) for poly(A)+ mRNA, random primers for broad coverage including rRNA/lncRNA, or gene‑specific primers for targeted assays.
• RNA input: Commonly 1 pg–5 µg total RNA per 10–20 µL reaction (optimize empirically; not item‑specific).
• RNase control: Work cold and nuclease‑free; include RNase inhibitor where compatible.
• gDNA contamination: Use no‑RT controls and, if appropriate, a DNase pre‑treatment step.
• Downstream compatibility: Dilute or heat‑inactivate the RT reaction per kit instructions (not provided here) before PCR/qPCR.

Performance validation

• Always include positive controls (validated RNA) and negative controls (no‑template, no‑RT) to assess specificity and contamination.

Reaction Conditions

Exact, item‑specific reaction conditions are not provided. The following are general literature guidelines for first‑strand cDNA synthesis; optimize empirically and consult the kit insert when available.

Typical ranges (general, not item‑specific)

• RNA input: 1 pg–5 µg total RNA or 1 pg–1 µg mRNA per 10–20 µL reaction.
• Primers: 50–500 ng oligo(dT)18–25, 50–200 ng random hexamers, or 0.1–1 µM gene‑specific primer per reaction.
• dNTPs: 0.2–1.0 mM each in the final reaction.
• Buffer/Mg2+: Use the supplied buffer; Mg2+ commonly 1–5 mM (kit‑dependent).
• Temperature/time: 37–55°C for 10–60 min for many RTs; higher temperatures (50–60°C) can improve structure resolution for GC‑rich RNA if the enzyme is thermostable. Heat‑inactivation step, if applicable, often 65–85°C for 5–10 min (enzyme‑dependent).
• Reaction volume: 10–50 µL typical.

Controls and QC

• Include no‑RT control to detect genomic DNA contamination and no‑template control to detect reagent contamination.
• Verify cDNA quality by amplifying a housekeeping gene spanning an exon–exon junction.

Compatibility

• Avoid carryover of ethanol, phenol, guanidinium, or heparin from RNA prep; these inhibit RT activity.

Note: Follow the specific instructions provided with this kit once available; the above values are general and not specifications for this item.

Safety and Handling

Safety information specific to this kit has not been provided in the Product Data.

Item‑specific safety

• GHS classification, signal word, H‑statements, and pictograms: Not specified for this item; refer to SDS.

General laboratory safety for nucleic acid/enzyme kits (not item‑specific)

• PPE: Wear lab coat, nitrile gloves, and safety glasses. Handle on ice to maintain performance; avoid skin/eye contact with solutions.
• Hygiene: Work in an RNase‑free area. Use dedicated, nuclease‑free consumables and aerosol‑resistant tips. Decontaminate benches and tools with RNase inactivating solutions.
• Incompatibilities: Avoid contamination with RNases, DNases, strong oxidizers, and microbial loads. Do not mix with bleach when guanidinium salts are present (if any; check SDS).
• First aid (general): In case of contact with eyes/skin, rinse with water for several minutes. If inhaled (aerosol), move to fresh air. If ingested, rinse mouth. Seek medical attention as per SDS guidance.
• Spill response: Absorb small spills with inert material; clean with RNase‑decontaminating solution, then water. Dispose according to institutional and local regulations.

Storage and transport

• Storage: Store at −20°C. Avoid repeated freezing and thawing (per Product Data). Aliquot enzyme solutions to minimize freeze–thaw cycles.
• Shipping: Shipped in ice chest with ice pads (per Product Data). Inspect upon arrival; keep cold.

Solvent Selection

This product is not a solvent; solvent selection per se is not applicable.

Practical considerations (general, for cDNA workflows)

• RNase‑free water: Use certified nuclease‑free water for RNA dilution and reaction setup. Avoid DEPC‑treated water in reactions that contain enzymatic activity unless verified compatible.
• Additives: If your downstream application requires specific ionic strength or co‑factors, use only those recommended in the kit insert (composition for this item is not specified).
• Sample matrix effects: Common RT inhibitors include carryover phenol, guanidinium salts, heparin, and ethanol. Ensure RNA purification is thorough and fully removes these.

Alternatives when adapting protocols

• If reactions must be concentrated or cleaned, use appropriate spin columns or magnetic beads rather than organic solvents, which can inactivate enzymes and degrade RNA.

Storage and Reconstitution

Item‑specific storage (from Product Data)

• Store at −20°C.
• Avoid repeated freezing and thawing.
• Shipped in ice chest with ice pads.

General best practices for this product type (not item‑specific)

• Aliquot enzyme‑containing solutions upon first use to minimize freeze–thaw cycles. Label aliquots with lot and date.
• Thaw on ice; mix gently by pipetting. Brief, low‑speed vortexing is acceptable for buffers; avoid vigorous agitation of enzyme vials.
• Keep components on ice during setup; return promptly to −20°C after use. Do not store in frost‑free freezers with warm cycles.
• If any component is lyophilized (not specified for this item), reconstitute with RNase‑free water or the supplied solvent per the product insert. Allow complete dissolution and gentle mixing; quick‑spin to collect.
• Avoid microbial or nuclease contamination: use nuclease‑free, sterile tubes and filtered aerosol‑resistant tips.

Stability and documentation

• For shelf life, allowable storage excursions, and component concentrations, consult the CoA/Spec Sheet and SDS.

Use restriction

• For research use only (per Product Data). Not for diagnostic procedures or therapeutic use.

Structure and Identity

This product is an enzymatic kit for first‑strand cDNA synthesis from RNA templates. It comprises multiple biological reagent components (e.g., enzyme(s), buffers, primers, and controls) rather than a single defined small molecule.

• SKU: H665509
• Product name: HiFiScript cDNA Synthesis Kit
• Category: Life Sciences research reagent (molecular biology kit)

Item‑specific identifiers

• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES / InChI / InChIKey / CAS / CID: Not applicable to a multi‑component kit; refer to component‑level SDS if needed.

Structural description (general)

• The functional activity derives from a reverse transcriptase (protein enzyme) and associated nucleic acid reagents. As proteins and oligonucleotides are macromolecules without a single repeatable small‑molecule structure, conventional small‑molecule descriptors (SMILES, InChI) do not apply.
• Typical kit architecture (general, literature):
  • Enzyme solution (reverse transcriptase in stabilizing buffer)
  • Reaction buffer(s) and dNTPs
  • Priming options (e.g., oligo(dT), random primers, or gene‑specific primers supplied by the user)
  • RNase‑free water

Notes

• For exact component list and concentrations, consult the product insert, CoA, and SDS for each vial.

Synthetic Utility

Although not a chemical synthesis reagent, the kit is highly useful in “synthetic biology” and molecular cloning workflows by converting RNA information into DNA that can be engineered.

General utilities (literature/general)

• cDNA cloning: Generate ORFs from mRNA for insertion into expression vectors.
• RACE and isoform mapping: Extend partial transcripts to full length for structural studies.
• Library construction: First‑strand cDNA is foundational for RNA‑seq and single‑cell transcriptomics libraries (when coupled with second‑strand synthesis and adapter ligation).
• Mutational analysis: Produce cDNA templates for targeted amplification and sequencing of splice junctions.

Retrosynthetic planning for gene constructs

• Starting material: Purified RNA of defined integrity (RIN/qi profile).
• Key transformation: RNA → DNA via reverse transcription under controlled priming and temperature conditions.
• Downstream transformations: PCR amplification, cloning, sequence verification, and expression testing.

Value to practitioners

• Enables stable DNA intermediates from labile RNA, facilitating archiving, quantitative assays, and manipulation with standard DNA tools.

Target Specificity

Not applicable. This product is a cDNA synthesis kit and does not contain an antibody or affinity reagent with defined antigen/epitope specificity.

• Tested targets, clone, isotype, or species reactivity: Not applicable.
• If using gene‑specific priming, target specificity is determined by the user‑supplied primer design and validation.

Foire aux questions

How should this product be stored?

Store at ?20 °C. Avoid repeated freeze–thaw cycles. Aliquot into single-use volumes before freezing.

How is this product shipped?

This product ships in an insulated container with ice pads. Unpack on arrival and transfer it to the storage condition stated above.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.