Qiagen, 150052, REPLI-g Cell WGA & WTA Kit (12)

REPLI-g SensiPhi DNA Polymerase, Buffers, and Reagents for parallel 12 x 60 µl whole genome amplification reactions and 12 x 60 µl whole transcriptome amplification reactions (typical yield: 20 µg from each reaction)

Features

- Parallel amplification of genomic DNA and RNA (total or mRNA-enriched)
- Easily correlate genomic status with transcription pattern
- Negligible sequence bias due to MDA technology
- Amplify gDNA and RNA directly from cells or tissue
- For use with all downstream applications, including NGS

Performance

Sample material (cells/total RNA): Research area
Cancer: Somatic genetic variant analysis
Tumor progression
Tumor stem cells/evolution
Tumor heterogeneity
Somatic genetic variant analysis
Human/animal: Biomarker research (SNPs, mutations, CNVs)
Mosaicism studies
Stem cell research
Genetic predisposition studies

Complete genomic and transcriptome coverage, with low experimental variability

Ideal for analyzing the genomic and transcriptomic profiles of limited samples

For use in a wide variety of 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 the procedure, the kits undergo stringent quality control to ensure complete functionality.
- The unique lysis buffer effectively stabilizes cellular RNA and DNA. This ensures that the resulting RNA accurately reflects the in vivo gene expression profile, and that RNA and DNA stays intact, maximizing genome coverage for subsequent analyses.
- All enzymatic steps have been specifically developed to enable efficient processing of RNA or DNA for accurate amplification. For example, these processes include effective gDNA removal prior to cDNA synthesis for RNA amplification and enzymatic DNA preparation that ensures efficient DNA amplification.
- Novel REPLI-g SensiPhi DNA Polymerase is used for Multiple Displacement Amplification (MDA). It is a newly developed, high-affinity enzyme that binds DNA more efficiently, especially when DNA concentration is low in the reaction mixture. In addition, and 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 DNA through stable hairpin structures that are resistant to Taq -based whole genome and whole transcriptome amplification procedures.

Unique components of the REPLI-g Cell WGA & WTA Kit

Procedure

- Lysis of cells : a sample (containing 25–1000 cells) is lysed efficiently within 5 minutes, with no effect on RNA or DNA integrity. Lysed cells are divided into two aliquots of equal volume. The first aliquot is used for WGA, while the second aliquot is used for WTA of total RNA, or alternatively, mRNA-enriched (poly A+) RNA.
- Whole genome amplification : following cell lysis, DNA from one of the aliquots is modified for high efficiency ligation. Due to the nature of the ligation reaction, DNA fragments are not assembled in the order in which they would have originally existed in the organism. However, this does not affect detection of nucleic acid sequences (e.g., polymorphisms) in downstream applications, such as NGS, array analysis, or qPCR. After ligation, amplification using MDA technology and the novel, high-affinity REPLI-g SensiPhi DNA Polymerase takes place.
- Whole transcriptome amplification : following cell lysis, gDNA is removed from the second aliquot prior to starting the WTA process, since accurate measurement of transcript levels depends on the elimination of false-positive results caused by gDNA contamination. cDNA is prepared for high efficiency ligation and amplification. Depending on the primer chosen during the subsequent reverse transcription reaction, all transcripts for total RNA enrichment or only polyadenylated transcripts for poly A+ mRNA enrichment will be amplified.

Applications

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

- Genomic DNA
- Mitochondrial DNA

- tRNAs and miRNAs
- Strongly degraded DNA or RNA

The REPLI-g Cell WGA & WTA Kit allows uniform parallel amplification of all transcripts and genomic regions from one very small sample. gDNA and cDNA amplified in parallel by the kit are highly suited for next-generation sequencing (both DNA-Seq and RNA-Seq*), analysis using arrays, genotyping applications, or quantitative PCR.

The REPLI-g Cell WGA & WTA Kit is suitable for amplification of:

Transcriptome (RNA) :

Genome (DNA) :

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