REPLI-g Cell WGA & WTA Kit
For parallel whole genome and whole transcriptome amplification from cells and limited samples
- 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
The REPLI-g Cell WGA & WTA Kit uniquely enables uniform whole genome amplification (WGA) and whole transcriptome amplification (WTA) in parallel reactions, allowing direct correlation of the genome with the transcriptome from very small samples (25–1000 cells). Dedicated buffers and reagents undergo a unique, controlled decontamination procedure to block amplification of contaminating nucleic acids by the REPLI-g method. The innovative lysis buffer effectively stabilizes cellular nucleic acids, ensuring accurate representation of both genomic DNA and RNA levels. Unbiased amplification of genomic DNA and total RNA or mRNA (poly A+) is achieved using innovative Multiple Displacement Amplification (MDA) technology and the novel REPLI-g SensiPhi DNA Polymerase. In contrast to PCR-based amplification technologies, high fidelity rates are increased up to 1000-fold, avoiding costly false positive or negative results.
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)
The REPLI-g Cell WGA & WTA Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
REPLI-g amplified cDNA and gDNA perform like gDNA in downstream experiments.|Multiple Displacement Amplification (MDA) technology.|High level of experimental reproducibility and minimized technical noise.|Highly suited for simultaneous genomic and transcriptomic analysis from the same limited sample.|REPLI-g Cell WGA & WTA Kit procedure.|
REPLI-g Kits amplify genomic DNA or RNA from a wide variety of sample types, generating amplified DNA and cDNA that performs just like gDNA and is highly suited for numerous downstream experiments.|Primers (arrows) anneal to the template DNA and are extended at 30ºC by REPLI-g SensiPhi DNA polymerase, which moves along the gDNA or cDNA template strand, displacing the complementary strand while becoming a template itself for replication. In contrast to PCR-based amplification, MDA does not require different temperatures and ends in very long fragments with low mutation rates.|Whole transcriptome amplification (WTA) using the REPLI-g Cell WGA & WTA Kit was performed using the mRNA (poly A+) enrichment protocol to reduce rRNA amplification. WTA amplified cDNA was fragmented (Covaris S220) and an NGS sequencing library was prepared using the GeneRead Library Prep I Kit (QIAGEN). Sequencing was done on a MiSeq Instrument (Illumina), RNA biotypes were mapped using Bowtie2, and reads per kilobase and million mapped reads (RPKM) were calculated. Results demonstrate comparable results, with an R2 value of 0.91, between the RPKM values of one 25-cell sample versus transcripts derived from the average of all WTA samples tested (5 samples of 25, 50, or 100 cells).|DNA and total RNA was amplified from the same 25-cell sample using the REPLI-g Cell WGA & WTA Kit. The same RT2 Profiler PCR Array was used to analyze 62 individual genes and their corresponding transcripts, which were sorted according their expression levels. All genes derived from genomic DNA were detected with CT levels between 20 and 30, while corresponding transcripts show variations due to different abundance, including undetectable transcript levels.|A sample (containing 25–1000 cells) is lysed efficiently within 5 minutes, with no effect on RNA integrity. Lysed cells are divided into two aliquots of equal volume, which can be processed in parallel. 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. Following cell lysis, (and gDNA removal and cDNA synthesis for the WTA process), cDNA from one of the aliquots is modified for high efficiency ligation. Amplification using MDA technology takes place using the novel, high-affinity REPLI-g SensiPhi DNA Polymerase.
Complete genomic and transcriptome coverage, with low experimental variability
The REPLI-g Cell WGA & WTA Kit contains the novel REPLI-g SensiPhi DNA Polymerase, as well as an optimized set of buffers and reagents for parallel whole genome amplification (WGA) and whole transcriptome amplification (WTA) from 25–1000 cells, or equivalently small samples. Following efficient cell lysis, (and complete removal of genomic DNA followed by sensitive reverse transcription for the WTA reaction), the kit utilizes Multiple Displacement Amplification (MDA) technology for complete and unbiased amplification of gDNA and cDNA (see figure Multiple Displacement Amplification (MDA) technology
). cDNA amplified using the REPLI-g Cell WGA & WTA Kit demonstrates a high degree of reproducibility from experiment to experiment, with minimal “technical noise”, ensuring highly reliable results (see figure High level of experimental reproducibility and minimized technical noise
). The amplified gDNA and cDNA can be easily used in a variety of downstream applications (see figure REPLI-g amplified cDNA and gDNA perform like gDNA in downstream experiments
Ideal for analyzing the genomic and transcriptomic profiles of limited samples
The REPLI-g Cell WGA & WTA Kit provides highly uniform, parallel amplification across the entire genome and transcriptome, with negligible sequence bias. Even from samples as low as 25 cells, the kit ensures reliable whole genome and transcriptome amplification, making it highly suited for analyzing the direct consequences of genotypic changes on transcriptomes (see figure Highly suited for simultaneous genomic and transcriptomic analysis from the same limited sample
For use in a wide variety of research areas
The REPLI-g Cell WGA & WTA Kit efficiently amplifies gDNA and cDNA directly from cells and can be used with various samples that are often analyzed in clinical research, including tumor cells, stem cells, sorted cells, or other small samples (see table).
||Somatic genetic variant analysis
|Tumor stem cells/evolution
|Somatic genetic variant analysis
|Biomarker research (SNPs, mutations, CNVs)
|Stem cell research
|Genetic predisposition studies
The REPLI-g Cell WGA & WTA Kit has been specifically designed to co-amplify genomic DNA and total RNA in parallel reactions from a very small sample, allowing direct correlation of the genome with the transcriptome. Regulation of transcription is driven by the genome in response to the environment. Mutations and variations in the genome, such as point mutations and copy number or structural variations, result in a variety of different genomes within species or within an organism (e.g., mosaic tissues and cancer). Some genomic variations may have a low influence on cellular or systemic regulation. Others can have a dramatic effect on transcription regulation and, ultimately, physiology. Because of the composite structure of tissues, analyzing the direct consequences of genotypic changes on transcriptomes can only be done with very small samples that are made up of identical cells.
The REPLI-g Cell WGA & WTA Kit provides highly uniform amplification across the entire genome and transcriptome, with negligible sequence bias. The kit uses Multiple Displacement Amplification (MDA) technology, which carries out isothermal gDNA/cDNA amplification utilizing a uniquely processive DNA polymerase capable of replicating up to 70 kb without dissociating from the DNA template (see figure Multiple Displacement Amplification (MDA) technology
Unique components of the REPLI-g Cell WGA & WTA Kit
- 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.
Genetic analyses often require large amounts of gDNA and cDNA, which can be challenging given the often small sample sizes. It is also not possible to obtain these when correlating the genomic status with the transcription pattern of just one very small sample. Parallel whole genome amplification and whole transcriptome amplification overcome the limits of low DNA or RNA quantity, allowing one sample of a small number of cells to be analyzed. The easy reaction setup, logical and streamlined processing steps, low handling time of just 20 minutes, and overall reaction time of only 4 hours for the complete, parallel amplification of gDNA and RNA, make the REPLI-g Cell WGA & WTA Kit procedure an easy and reliable method to use.
The REPLI-g Cell WGA & WTA Kit contains reagents for the following sequential reactions (see figure 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.
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:
- mRNA with poly A+ tail
- Total RNA
- All regions of RNA transcripts
- 3’ ends of mRNAs
- Lnc and linc RNAs
It is not suitable for use with small nucleic acids (for example)
- Genomic DNA
- Mitochondrial DNA
* If performing next-generation sequencing, be aware that more than 90% of reads are derived from rRNA; therefore, sufficient reads must be obtained when performing whole transcriptome sequencing following amplification of total RNA. Amplification of rRNA does not occur when amplifying mRNA-enriched (poly A+) RNA by omitting the random primers from the reaction.
- tRNAs and miRNAs
- Strongly degraded DNA or RNA