Search
Search
View additional product information for SuperScript™ IV Single Cell/Low Input cDNA PreAmp Kit - FAQs (11752192, 11752048, 11752384, 11752096, 11752480)
15 product FAQs found
We recommend using TaqMan Fast Advanced Master Mix for all TaqMan‑based detection methods.
We recommend using PowerTrack SYBR Green Master Mix for all SYBR Green‑based detection methods.
Cycle number depends on sample type and input amount.
For example:
- 21 cycles for 2 pg of total RNA
- 18 cycles for 10 pg of total RNA, or single cells
- 15 cycles for 100 pg of total RNA, or up to 10 cells
- 11 cycles for 1-10 ng of total RNA, or 100–1,000 cells
- RNA quantity in cells can vary by cell type, cell cycle, and cell health. Optimization of the PCR cycle number may be needed to reach desired yields. An additional 2–3 cycles are required for smaller cells, such as Jurkat, Daudi, or peripheral blood mononuclear cells (PBMC).
The master mix includes Platinum SuperFi U DNA Polymerase. It provides the same benefits as Platinum SuperFi II DNA Polymerase, but is also compatible with uracil.
Lysis buffer is included in the kit, therefore intact mammalian cells can be used. Purification of total RNA is required for cDNA preamplification of RNA isolated from plant, fungi, or other cell wall‑containing organisms.
rRNA depletion or mRNA enrichment is not required, because only poly(A)‑containing RNA is amplified.
The SuperScript IVSingle Cell/Low Input cDNA PreAmp Kt has only been tested with Agencourt AMPure XP beads. Therefore, we cannot recommend any alternatives.
We recommend using Agencourt AMPure XP beads (Beckman Coulter A63880) according to the protocol provided in the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit’s User Guide.
For downstream analysis by (next-generation sequencing) NGS, preamplified cDNA should be purified to remove residual primers, nucleotides and enzymes that may impact library preparation. For product analysis by qPCR, purification is not required but can be used to concentrate samples for detection of low-expression targets. For unpurified cDNA samples, samples should be diluted 1:10 or 1:100, prior qPCR. For purified cDNA samples, dilution is not necessary. The purification step is also recommended prior to storing preamplified cDNA at -20 degrees C for more than 1 week.
TaqMan PreAmp Master Mix is designed for targeted preamplification of up to 100 targets from small amounts of cDNA, while SuperScript IV Single Cell/Low Input cDNA PreAmp Kit allows global preamplification of full-length cDNA from single cells or low input RNA. It provides reagents for cell lysis, reverse transcription and preamplification steps.
This kit is designed for global full-length cDNA synthesis and preamplification. However, in downstream analysis, either whole transcriptome or specific targets of interest can be analyzed. It is compatible with downstream analysis by both next-generation sequencing (NGS) and real-time PCR.
This kit is designed for global full-length cDNA preamplification. It leverages SMART technology utilizing oligo (dT)-based priming. Therefore, to obtain full length cDNA, we recommend high quality intact poly(A) RNA.
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.