A team led by researchers at the University of Florida present a single-cell RNA amplification approach combining exponential and linear amplification steps using a limited number of PCR cycles and T7-driven in vitro transcription (IVT). Thier approach allows for unlimited RNA amplification and enables detection of gene isoforms, non-coding RNAs, and mRNAs with short or absent poly(A) tails.
By introducing major technical modifications, they have developed an RNA amplification method that is suitable for single-cell RNA amplification and significantly diminishes the problem of 3′bias. The major modifications that distinguish this new protocol include:
- Introduction of “extending primers” that contain random and semi-random sequences at the 3′ ends during PCR allows tagging of 3′ ends of cDNAs. The design of one of the extending primers includes a Kozak sequence at its 3′ ends that allows capture of 5′ ends of a gene’s coding sequence.
- The combination of modified oligo(dT) and modified random primers allows a decrease of the size distribution of the resulting cDNA fragments, making the PCR step more efficient and consequently improving the preservation of relative gene abundance.
- Detection of all parts of transcripts without 3′ bias, including the recognition of isoforms and mRNAs with short or completely absent poly(A) tails, as well as the identification of noncoding RNAs. The priming approach that utilizes the combination of oligo(dT) and random primers during the reverse transcription (RT) reaction is superior to any other priming strategy, and it secures full-length RNA coverage (18).
- Carryover of reverse transcriptase results in PCR inhibition, which can become significantly pronounced when minute amounts of RNA are used (19–24). This approach has greatly diminished this reverse transcriptase inhibitory effect on the following PCR step.
- This method generates 200–250 μg of amplified RNA from a single cell, sufficient to apply to virtually any RNA technique.
- Cost is dramatically reduced (up to 5-fold) in comparison to commercially available kits. This is relevant because single-cell experiments usually require simultaneous study of at least 50–100 individual cells.
RNA amplification scheme