Invitrogen™ SuperScript™ IV CellsDirect™ cDNA Synthesis Kit

Catalog No.	No. of Reactions
11-750-150	50 Reactions
11-750-350	500 Reactions

Catalog No. 11-750-150 Supplier Invitrogen™ Supplier No. 11750150

Description

The SuperScript IV CellsDirect cDNA Synthesis Kit is optimized for the synthesis of first-strand cDNA directly from a mammalian cell lysate without first isolating RNA. Cell lysis and reverse transcription are performed in the same tube, and the resulting first-strand cDNA is ready to use in PCR and qPCR.

The SuperScript IV CellsDirect cDNA Synthesis Kit is optimized for the synthesis of first-strand cDNA directly from a mammalian cell lysate without first isolating RNA. Cell lysis and reverse transcription are performed in the same tube, and the resulting first-strand cDNA is ready to use in PCR and qPCR.

Features of the SuperScript IV CellsDirect Synthesis Kit
• Compatible with a wide range of mammalian cell types grown under different treatment conditions
• Single-tube format minimizes reagent loss, sample loss, and handling time
• Total lysate volume can be used in first-strand cDNA synthesis reaction, providing greater yields with a limited number of cells and allowing for detection of rare transcripts
• High-quality cDNA generated for use in a variety of applications, including PCR and qPCR
• Simple protocol takes approximately 37 minutes
• Compatible with TaqMan and SYBR Green qPCR master mixes from various manufacturers
• Optimized performance for 1–10,000 cells per sample, results equivalent to those from purified RNA

Features of the SuperScript IV Reverse Transcriptase included in this kit
• Reduced RNase H activity
• High thermal stability
• High yields of cDNA in the first-strand synthesis reaction for greater sensitivity and enhanced detection of rare transcripts
• Superior tolerance to various RT inhibitors

SuperScript IV reverse transcriptase is a top choice for all RT-PCR and qRT-PCR applications. It is a proprietary MMLV mutant with superior robustness and reliability in RT reactions. It is significantly improved over SuperScript III reverse transcriptase in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of the previous enzyme, including increased thermostability, highly efficient full-length cDNA synthesis, and reduced RNase activity.

How it works
In traditional RT-PCR, RNA is first isolated from cells in a time-consuming procedure that can lead to a loss of material. Using the SuperScript IV CellsDirect cDNA Synthesis Kit, the cells are lysed and the cDNA is generated from the lysate in a single tube with minimal handling and no sample loss. DNase I treatment is an optional step and can be used to eliminate genomic DNA prior to first-strand synthesis. This kit has been optimized for small cell samples, ranging from 10,000 cells down to a single cell.

Specifications

• Content And Storage:

  • SuperScript IV CellsDirect Lysis Solution (2 x 1.25 mL)
  • SuperScript IV CellsDirect Stop Solution (1 x 250 μL)
  • Lysis Enhancer (1 x 25 μL)
  • Dnase I (1 x 55 μL)
  • SuperScript IV RT Master Mix (1 x 400 μL)
  • SuperScript IV No RT Control (1 x 400 μL)
  
  Store at –20°C.
  
  

• Format: Kit
• GC-Rich PCR Performance: High
• Reaction Speed: 10 min.
• Technique: Reverse Transcription
• Optimal Reaction Temperature: 50°C
• Quantity: 50 reactions
• Reverse Transcriptase: SuperScript IV
• Ribonuclease H Activity: Reduced
• Shipping Condition: Dry Ice
• For Use With (Application): RT-PCR, Real Time PCR (qPCR)
• Final Product Type: First-Strand cDNA
• No. of Reactions: 50 Reactions
• Reaction Format: Premixed Components
• Reagent Type: Reverse Transcription
• Starting Material: Cells

Frequently Asked Questions (FAQs)

What are the RT primers included in the SuperScript IV CellsDirect cDNA Synthesis Kit (Cat. No. 11750150, 11750350)?

The RT master mix contains both random and oligo dT primers.

What primers come in the SuperScript IV CellsDirect cDNA Synthesis Kit (Cat. No. 11750150)? Is it Oligo(dT) primers or random hexamers?

It is both. The RT master mix includes both, random hexamers and Oligo(dT) primers.

In your SuperScript IV RT protocols, there is no ezDNase inactivation step. Will ezDNase be active to affect RNA or the RT reaction?

The Invitrogen ezDNase Enzyme is a novel DNase that is highly specific for double-stranded DNA. It has no activity on single-stranded DNA in RT reactions (primers or probes), or on RNA. The enzyme is also thermolabile—it is inactivated quickly at temperatures typical for the SuperScript IV RT reaction (e.g., 50 degrees C). The additional inactivation step is therefore not required for most RT-qPCR applications. If the RNA sample is to be used for RT-PCR of fragments ≥3 kb, incubate the sample for 5 minutes at 55 degrees C in the presence of 10 mM DTT to inactivate the enzyme.

Which SuperScript IV RT format do you recommend for real-time PCR applications?

For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix. The cDNA synthesis reaction setup with this master mix requires fewer pipetting steps and therefore reduces variation in the data. SuperScript IV RT, as a component of the master mix, offers the highest efficiency of cDNA synthesis step compared to competitors’ products.

Are there any significant changes in the SuperScript IV RT protocol compared to the SuperScript III RT protocol?

The only difference is that the incubation time for the reverse transcription reaction has been reduced to 10 minutes and inactivation time has been reduced to 5 minutes at 85 degrees C.

Can I get comparable cDNA yield and length using the SuperScript IV RT 10-minute protocol as when using the 50-minute protocol for SuperScript III RT?

When compared with SuperScript III RT (and other manufacturers’ RTs) in a synthesis reaction for a 9 kb cDNA, SuperScript IV RT performed successful synthesis in just 10 minutes and did so with comparable (or improved) yield (as shown by gel band density).

If I run out of Lysis Solution in the SuperScript IV CellsDirect cDNA Synthesis Kit, can I use my home-brewed lysis solution or lysis solution from another kit?

All the reagents in the SuperScript IV CellsDirect cDNA Synthesis Kit have been tested and optimized for the direct RT protocol. We recommend using only the reagents offered in the kit and following the provided protocol. Efficiency could be compromised when using a lysis solution from an external source. Additional SuperScript IV CellsDirect Lysis Reagents can be ordered separately (Cat. No. 11750550).

What type of SuperScript IV master mix is used in the SuperScript IV CellsDirect cDNA Synthesis Kit? Can I subsitute it with the SuperScript IV VILO master mix?

The SuperScript IV master mix in the SuperScript IV CellsDirect cDNA Synthesis Kit is specifically designed and optimized in the direct RT protocol. We don't recommend using the SuperScript IV VILO master mix to replace the SuperScript IV master mix supplied in the kit.

In the SuperScript IV CellsDirect cDNA Synthesis Kit protocol, can I skip the Stop Solution step?

We recommend following all the steps recommended in the SuperScript IV CellsDirect cDNA Synthesis Kit protocol to ensure optimal, reproducible results. The Stop Solution step after cell lysis is necessary for complete inhibition of enzymes used in the lysis step and therefore for effective subsequent reactions (RT and PCR). The stop step cannot be skipped if the Lysis Enhancer and DNase I are used. To put it simply, if the Lysis Enhancer remains active, it can hydrolyze SuperScript IV RT and other proteins in RT-PCR. If DNase I remains active, it can hydrolyze PCR products.

When using the SuperScript IV CellsDirect cDNA Synthesis Kit, what control should I use for lysis/reverse transcription (RT) steps?

As a positive control for lysis/RT reactions, we would recommend using the Xeno RNA Control from the SYBR Green Cells-to-CT Control Kit (Cat. No. 4402959) that includes specific PCR primers for SYBR Green assays or from the TaqMan Cells-to-CT Control Kit (Cat. No. 4386995) that includes PCR primers and probes for TaqMan assays.

How long does the SuperScript IV CellsDirect cDNA Synthesis Kit protocol take?

The protocol takes about 35 min, including lysis and reverse transcription (RT) steps.

When using the SuperScript IV CellsDirect cDNA Synthesis Kit, do I need to treat my samples with DNase I every time?

We would recommend treating samples with DNase I if you are using higher than 100 cells per sample. If you are using 1-100 cells per sample, adding DNase (and Lysis Enhancer) into the Lysis Solution is optional. In that case, use only the recommended amount of Lysis Solution, incubate for 7 min at room temperature, and do not use Stop Solution. For the reverse transcription reaction, adjust the volume with nuclease-free water.

Can I purchase DNAse I from the SuperScript IV CellsDirect cDNA Synthesis Kit as a stand-alone item?

Yes, you can purchase DNase I (Cat. No. 18047019) separately.

For the SuperScript IV CellsDirect cDNA Synthesis Kit, I have to start with more than 10,000 cells. What is your recommendation?

For sample cell numbers greater than 10,000, we would recommend using adjusted volumes of reagents for the lysis reaction. We tested different combinations of reagents and observed that for high-density cell samples, the better option is to use higher reaction volumes in the lysis step. For further details, go to the SuperScript IV CellsDirect cDNA Synthesis Kit User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0019059_SS_IV_CellsDirectcDNA_Kit_UG.pdf) and refer to the section "Recommended volumes for high-density cell samples" on page 20.

Which end-point PCR reagents do you recommend using with the SuperScript IV CellsDirect cDNA Synthesis Kit?

For end-point PCR analysis with the SuperScript IV CellsDirect cDNA Synthesis Kit, we recommend using the Platinum SuperFi II PCR Master Mix (2X)/Platinum SuperFi II Green PCR Master Mix (2X) (Cat. No. 12368010, 12369010) or Platinum II Hot-Start PCR Master Mix (2X)/Platinum II Hot-Start Green PCR Master Mix (2X) (Cat. No. 14000012, 14001012).

Is it possible to use the SuperScript IV CellsDirect cDNA Synthesis Kit with frozen cells?

Yes, however, we recommend ensuring that cells are stored at -70 degrees C and that all media is removed before proceeding to the cell lysis step

Can I purchase lysis reagents from the SuperScript IV CellsDirect cDNA Synthesis Kit as stand-alone items?

You can purchase SuperScript IV CellsDirect Lysis Reagents (Cat. No. 11750550) separately. This kit includes the SuperScript IV CellsDirect Lysis Solution, SuperScript IV CellsDirect Stop Solution, and Lysis Enhancer.

What steps should I take to prevent RNA degradation when using the SuperScript IV CellsDirect cDNA Synthesis Kit?

We recommend keeping the cells on ice before starting the cell lysis procedure. Remove the cells from ice only after the Lysis Solution is added. To monitor RNA degradation, we recommend adding an RNA positive control in the RT reaction.

When using the SuperScript IV CellsDirect cDNA Synthesis Kit, how can I prevent nuclease contamination?

To prevent nuclease contamination, RNase inhibitor is included in the kit composition. For additional prevention of nuclease contamination, we recommend the following:
- Wear laboratory gloves during the procedures.
- Use nucleic acid-free pipette tips to handle the reagents and avoid putting used tips into the reagent containers.
- Decontaminate lab benches and pipettes before you begin.
- Close tube caps carefully and slowly and always keep them closed after every use.

What is the recommended storage temperature for the components of the SuperScript IV CellsDirect cDNA Synthesis Kit?

We recommend storing the SuperScript IV CellsDirect Stop Solution, DNase I, Lysis Enhancer, SuperScript IV RT Master Mix, SuperScript IV No RT Control at -20 degrees C, whereas the SuperScript IV CellsDirect Lysis Solution should be stored at 4 degrees C.

When using the SuperScript IV CellsDirect cDNA Synthesis Kit, what kind of primers should I choose for the reverse transcription (RT) reaction?

With the SuperScript IV CellsDirect cDNA Synthesis Kit, there is no need to add additional primers. A mix of oligo(dT) primers and random primers is already provided in the SuperScript IV RT Master Mix.

I am studying genes that have low expression levels that I can hardly detect in cells. Can I use the SuperScript IV CellsDirect cDNA Synthesis Kit for detection of low-expression genes?

Yes, the SuperScript IV CellsDirect cDNA Synthesis Kit is a good choice for detection of low-expression genes. The kit contains SuperScript IV Reverse Transcriptase which has significantly improved resistance to a variety of inhibitors that can interfere with cDNA synthesis, and significantly better processivity. These features of SuperScript IV Reverse Transcriptase allow sensitive detection of genes that have low expression levels.
For qPCR analysis with the SuperScript IV CellsDirect cDNA Synthesis Kit, we recommend using the PowerTrack SYBR Green Master Mix (Cat. No. A46012) for SYBR Green assays and the TaqMan Fast Advanced Master Mix (Cat. No. 4444556) for TaqMan assays.
For better detection sensitivity in qPCR, you can try adding more cDNA into the qPCR reaction following the cDNA input recommendations in the respective manual.

I have to perform RNA detection from a single cell with a small amount of testing material. Can I use the SuperScript IV CellsDirect cDNA Synthesis Kit? Will I get enough cDNA for downstream end-point PCR or qPCR analysis?

With the SuperScript IV CellsDirect cDNA Synthesis Kit, every reaction occurs in a single tube. This feature minimizes reagent loss, sample loss, and handling time. Additionally, the entire lysate volume can be used in the first-strand cDNA synthesis reaction, providing sufficient testing material for single cell applications. For cDNA analysis, use our recommended qPCR and end-point PCR master mixes for high detection sensitivity, as shown below:
For qPCR analysis, we recommend using the PowerTrack SYBR Green Master Mix (Cat. No. A46012) for SYBR Green assays and the TaqMan Fast Advanced Master Mix (Cat. No. 4444556) for TaqMan assays.
For end-point PCR analysis, we recommend using the Platinum SuperFi II PCR Master Mix (2X)/Platinum SuperFi II Green PCR Master Mix (2X) (Cat. Nos. 12368010 and 12369010) or Platinum II Hot-Start PCR Master Mix (2X)/Platinum II Hot-Start Green PCR Master Mix (2X) (Cat. Nos. 14000012 and 14001012).
Alternatively, consider using the SuperScript IV Single Cell/Low-Input cDNA PreAmp kit providing cDNA preamplification reagent for single cell analysis. Learn more at thermofisher.com/directrt.

When using the SuperScript IV CellsDirect cDNA Synthesis Kit, how should I remove gDNA from the final reaction?

For removing gDNA from the final reaction we recommend the following:
- Ensure DNase I is added to the Lysis Solution and the incubation is performed as described.
- Ensure the lysis reagents are kept on ice throughout the procedure.
- Ensure the lysis reaction is performed at room temperature.
- Use exon‑exon junction primers that are cDNA‑specific and do not bind to genomic DNA.
- Make sure that all media is removed from the wells/tube (cells).

Which cell lines have been tested to work with the SuperScript IV CellsDirect cDNA Synthesis Kit?

Here is a list of cell lines that have been tested to work with the SuperScript IV CellsDirect cDNA Synthesis Kit:

Cell line; Culture properties; Organism; Tissue
- HeLa; adherent; Homo sapiens; cervical adenocarcinoma
- HeLa S3; suspension; Homo sapiens; cervical adenocarcinoma
- Raji; suspension; Homo sapiens; B lymphocyte
- NIH/3T3; adherent; Mus Musculus (mouse); embryonic fibroblast
- HEK-293; adherent; Homo sapiens; kidney
- Jurkat; suspension; Homo sapiens; acute T cell leukemia
- Daudi; suspension; Homo sapiens; Burkitt's lymphoma
- K562; suspension; Homo sapiens; bone marrow
- iPSC; adherent; Homo sapiens; stem cells
- Balb/3T3; adherent; Mus Musculus (mouse); embryonic fibroblast

Can I use the SuperScript IV CellsDirect cDNA Synthesis Kit for RNA detection from adherent cells?

Yes, the SuperScript IV CellsDirect cDNA Synthesis Kit can be used with different types of cultured cells: adherent, suspension, and FACS-sorted cells.

Which qPCR reagents do you recommend using with the SuperScript IV CellsDirect cDNA Synthesis Kit?

For qPCR analysis with the SuperScript IV CellsDirect cDNA Synthesis Kit, we recommend using the PowerTrack SYBR Green Master Mix (Cat. No. A46012) for SYBR Green assays and the TaqMan Fast Advanced Master Mix (Cat. No. 4444556) for TaqMan assays.

Are qPCR reagents included with the SuperScript IV CellsDirect cDNA Synthesis Kit?

No, you need to order qPCR reagents separately.

What is the minimum amount of mammalian cells I can use in the lysis step with the SuperScript IV CellsDirect cDNA Synthesis Kit?

With the SuperScript IV CellsDirect cDNA Synthesis Kit, you can detect RNA from single cells up to 10,000 cells per sample with results equivalent to those from purified RNA.

Safety and Handling

WARNING: Cancer - www.P65Warnings.ca.gov