Search
Search
View additional product information for SuperScript™ IV CellsDirect™ cDNA Synthesis Kit - FAQs (11750350, 11750150)
29 product FAQs found
The RT master mix contains both random and oligo dT primers.
It is both. The RT master mix includes both, random hexamers and Oligo(dT) primers.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
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°C). The additional inactivation step is therefore not required in RT-qPCR applications.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix (Cat. No. 11756050). 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.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
The only change is that the incubation time for the reverse transcription reaction has been reduced from 50 minutes to 10 minutes. All the other parameters and steps are the same.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
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).
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
All the reagents in the SuperScript IV CellsDirect cDNA Synthesis Kit have been tested and optimized for the direct RT protocol. It's important to use only reagents offered in the kit and follow the protocol. Efficiency could be compromised if lysis solution from other sources is used.
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.
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.
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.
The protocol takes about 35 min, including lysis and reverse transcription (RT) steps.
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.
Yes, you can purchase DNase I (Cat. No. 18047019) separately.
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.
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).
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
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.
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.
For prevention of nuclease contamination we would 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.
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.
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.
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.
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 further 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. 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).
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).
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
Yes, the SuperScript IV CellsDirect cDNA Synthesis Kit can be used with different types of cultured cells: adherent, suspension, and FACS-sorted cells.
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.
No, you need to order qPCR reagents separately.
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.