A novel global cDNA amplification technology directly from an RNA template via reverse transcription

We developed a method, Reverse Transcription with Random Displacement Amplification (RT-RamDA), to reverse transcribe and amplify cDNA directly from RNA templates using strand displacement and RNA-dependent DNA polymerase activity. This method resulted in a cDNA yield approximately 100-fold higher than conventional RT with high sensitivity, enabling gene expression profiling of low-expression genes with a small amount of RNA, such as that obtained at the single-cell level. Overall design: 12 samples purified total RNA samples diluted to 10 pg were sequenced by using RT-RamDA and DNA SMART ChIP-Seq Kit. RT-RamDA was performed using different conditions of reverse transcription primer, oligo (dT), N6, not-so-random (NSR) primers which developed to reduce the synthesis of cDNAs derived from ribosomal RNAs with random priming (33), N6/oligo (dT) and NSR/oligo (dT). Moreover, 3 rRNA-depleted samples derived from 200 ng of total RNA were sequenced by using KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems).