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.
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.
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).
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.