Qiagen, 150063, REPLI-g WTA Single Cell Kit (24)

REPLI-g SensiPhi DNA Polymerase, Buffers, and Reagents for 24 x 60 µl whole transcriptome amplification reactions (typical yield: 20 µg)

Features

- Complete transcriptome coverage from just single cells
- Uniform WTA with negligible sequence bias due to MDA technology
- Optimized for use with new technologies, including NGS
- Amplification of total RNA or mRNA-enriched (poly A+) RNA
- Novel tool for cancer and stem cell research

Performance

Sample material (cells/total RNA): Research area
Human/animal: Biomarker research (expression)
Stem cell research
Analysis of circulating fetal cells
Mosaicism studies
Genetic predisposition studies
Typing of transgenic animals
Cancer: Somatic genetic variant analysis
Tumor progression
Tumor stem cells/evoluation
Analysis of circulating tumor cells

Complete transcriptome coverage, with low experimental variability

Significant number of reads map to protein-coding RNA

Reliable detection of low-abundance transcripts

For use in a wide variety of applications and research areas

Principle

- All of the kit’s enzymes and amplification components undergo a unique, controlled decontamination procedure to ensure elimination of REPLI‑g amplifiable contaminating DNA or RNA. Following this process, the kits undergo stringent quality control to ensure complete functionality.
- The innovative lysis buffer effectively stabilizes cellular RNA. This ensures that the resulting RNA accurately reflects the in vivo gene expression profile.
- All enzymatic steps have been specifically developed to enable efficient processing of RNA for accurate amplification. For example, these processes include effective gDNA removal prior to cDNA synthesis.
- Novel REPLI-g SensiPhi DNA Polymerase is used for Multiple Displacement Amplification (MDA). It is a newly developed, high-affinity enzyme that binds cDNA more efficiently, especially when the cDNA concentration is low in the reaction mixture. In addition, in contrast to PCR-based methods, REPLI-g SensiPhi DNA Polymerase has strong proofreading activity that results in 1000-fold fewer errors. It also has strong strand-displacement activity, enabling replication of cDNA through stable hairpin structures that are resistant to Taq -based whole genome or whole transcriptome amplification procedures.

Unique components of the REPLI-g WTA Single Cell Kit

Procedure

- Lysis of cells : a single cell sample (containing 1–1000 cells) is lysed efficiently within 5 minutes, with no effect on RNA integrity. The lysed sample is used for WTA of total RNA or, optionally, mRNA-enriched (poly A+) RNA.
- Generation of cDNA : following cell lysis, gDNA is removed prior to the WTA process, since accurate measurement of transcript levels depends on the elimination of false-positive results caused by gDNA contamination. Depending on the primer chosen during the subsequent reverse transcription reaction, all transcripts (if performing total RNA enrichment using random and oligo dT primers) or only poly-adenylated transcripts (if performing poly A+ mRNA enrichment using oligo dT primers) will be amplified. Consequently, the reaction will contain a mixture of random and oligo dT primers, or oligo dT primers only, which reduces rRNA amplification and ensures the 3’ ends of cDNA are reverse transcribed (transcript sizes are approximately 700–1000 bp).
- Ligation : the synthesized cDNA is ligated using a high-efficiency ligation mix. Due to the nature of the subsequent ligation reaction, cDNA fragments are not assembled in the order in which they would have originally existed in the cell. However, this does not affect the detection of nucleic acid sequences, such as splicing regions, in downstream applications like NGS or qPCR.
- Whole transcriptome amplification : The ligated cDNA is amplified utilizing MDA technology, with the novel REPLI-g SensiPhi DNA Polymerase, in an isothermal reaction lasting 2 hours.

- The protocol “Amplification of the 3’ Regions of mRNA (Poly A+) from Single Cells” amplifies mRNAs (and other RNAs) with poly A+ tails only and is highly suited for a wide range of applications, including NGS (RNA-Seq), real-time PCR, and microarray analysis.
- The protocol “Amplification of Total RNA from Single Cells” amplifies the complete transcriptome, including RNAs with and without poly A+ tails, lnc RNAs, and linc RNAs. Note that rRNA is also amplified and will be present at a high level following amplification. If working with sequence-specific probes, such as with qPCR or arrays, the amplified rRNA will not affect downstream application results. If using the amplified RNA for RNA-Seq, be aware that more than 90% of reads are derived from rRNA; therefore sufficient reads must be obtained when performing whole-transcriptome sequencing.
- The protocol “Amplification of Purified RNA” is optimized for whole transcriptome amplification from total or enriched RNA templates and is highly suited for a wide range of applications, including NGS, real-time PCR, and microarray analysis.

Applications

- mRNA with poly A+ tails
- Total RNA
- All regions of RNA transcripts
- 3’ ends of mRNAs
- Lnc and linc RNAs

- tRNAs and miRNAs

The REPLI-g WTA Single Cell Kit allows uniform amplification of all transcripts from very small samples, accurately representing the transcription pattern of a single cell with very limited, or no, amplification bias. Depending on the protocol, amplified cDNA is highly suited for use in next-generation sequencing (RNA-Seq), gene expression arrays, or for quantitative PCR analysis.

The REPLI-g WTA Single Cell Kit can be used for whole transcriptome amplification for analysis of:

It is not suitable for use with small nucleic acids (for example):