A single cell sample (containing 1–1000 cells) is lysed efficiently within 5 minutes, with no effect on RNA integrity. Following cell lysis, gDNA is removed prior to the WTA process. 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. The synthesized cDNA is ligated in a high-efficiency ligation reaction. Ligated cDNA is then amplified utilizing MDA technology with the novel REPLI-g SensiPhi DNA Polymerase in an isothermal reaction lasting 2 hours.
Primers (arrows) anneal to the template DNA and are extended at 30ºC by REPLI-g SensiPhi DNA Polymerase, which moves along the 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.
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.
Whole transcriptome amplification was performed from 15 different single cells or 15 replicates of 10 pg total RNA using the REPLI-g WTA Single Cell Kit. Real-time PCR analysis of 1 ng WTA-amplified cDNA from a variety of different transcripts was done to quantify high-, medium-, and low-copy transcripts. [A] Box plots were calculated from ΔCT (CT [WTA-DNA] – average CT [WTA-DNA]) to differentiate biological differences from the method-derived technical noise. The green box indicates increased gene expression and the red box indicates decreased gene expression compared to the mean. Technical noise, representing the limit of sensitivity, is depicted by the white box, which is clearly less than the biological differences detected. [B] Analysis of a low-copy transcript (abl-1) in 15 individual cells demonstrates the variability of transcript levels between individual cells and that most CT values are outside the low level of technical noise (depicted by black lines). These findings indicate that the REPLI-g WTA Single Cell Kit is highly suited to analyze even low-abundance transcripts from just single cells.
Whole transcriptome amplification was performed using 20 pg of total RNA. [A] Unlike kits from other suppliers, which were less successful at amplifying the same amount of the same transcript, 100% of high-, medium-, and low-abundance transcripts were detected following RNA amplification using the REPLI-g WTA Single Cell Kit. [B] Real–time PCR of 22 medium-abundance transcripts, representing the medium-abundance transcript row in [A], demonstrated that only the REPLI-g WTA Single Cell Kit reliably amplified all transcripts. Transcripts that could not be detected gave CT values >40 (red bar).
[A] REPLI-g WTA Single Cell reactions were performed using the mRNA (poly A+) enrichment protocol to reduce rRNA amplification. WTA amplified cDNA was prepared as described in "High number of mappable reads from just 3 cells" and sequenced on a MiSeq Instrument (Illumina). RNA biotypes were mapped to single-transcript RNA using Bowtie2 and reads per kilobase and million mapped reads (RPKM) were calculated. Results demonstrate comparable average RPKM values of the 3-cell samples versus transcripts derived from WTA samples (10–50 cells). [B] REPLI-g WTA Single Cell reactions, including rRNA amplification, were performed on individual human cells. Real-time PCR of various transcripts (18S rRNA, 28S rRNA, ddx5, beta-actin, HPRT, GAPDH, PPIA, c-myc, RPS27a, BANF-1, abl-1) was done using QuantiFast SYBR Green PCR reagents and 1 ng of WTA-cDNA. CT values normalized to 18S rRNA from two individual single cell WTA reactions demonstrate a high level of concordance in RNA amplification between experiments, with a high R2 value of >0.97.
REPLI-g WTA Single Cell reactions were performed on 3–1000 cells in various replicates, 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) and RNA biotypes were mapped using Bowtie2. Results demonstrate that the majority of reads (>80%) are mappable to protein coding RNA and Linc RNAs, and that a negligible number of reads map to other, non-targeted RNA biotypes (data for minor RNA biotypes not shown). Comparable results between values obtained after the sequencing of all WTA samples (Mean value [all cells]) and 3-cell WTA samples (Mean value [3 cells]) were obtained.