Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium.
In addition to messenger RNA (mRNA), noncoding RNAs (ncRNAs) are essential components in cellular machineries for translation and splicing. Besides their housekeeping functions, ncRNAs are involved in cell type-specific regulation of translation, mRNA stability, genome structure, and accessibility. To have a comprehensive understanding of the identities and functions of different cell types, a method to comprehensively quantify both mRNA and ncRNA in a sensitive manner is highly desirable.



Here we tried to develop a system capable of concurrently profiling both mRNA and ncRNA by polyadenylating RNA in samples before reverse transcription. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. The single-tube amplification (STA) system was applied to single to 100 cells of 293T cells, human pluripotent stem cells (hPSCs) and their differentiated endothelial progenies to validate its quantitative power and sensitivity by qPCR and high-throughput sequencing.



Using microRNA (miRNA) as an example, we showed that complementary DNA (cDNA) from ncRNAs could be amplified and specifically detected from a few cells within a single tube. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. With 100 human embryonic stem cells (hESCs) and their differentiated endothelial cells as input for high-throughput sequencing, the single-tube amplification (STA) system revealed both well-known and other miRNAs selectively enriched in each cell type. The selective enrichment of the miRNAs was further verified by qPCR with 293FT cells and a human induced pluripotent stem cell (hiPSC) line. In addition, the detection of other non-miRNA transcripts indicated that the STA target was not limited to miRNA, but extended to other ncRNAs and mRNAs as well. Finally, the STA system was capable of detecting miRNA and mRNA expression down to single cells, albeit with some loss of sensitivity and power.



Overall, STA offered a simple and sensitive way to concurrently quantify both mRNA and ncRNA expression in low-cell-number samples for both qPCR and high-throughput sequencing.
Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan.
Yi-Hsuan
YH
Lee
Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, No. 35, Xiaodong Rd, Tainan, 70457, Taiwan.
Ya-Wen
YW
Hsueh
Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, No. 35, Xiaodong Rd, Tainan, 70457, Taiwan.
Yao-Hung
YH
Peng
Department of Pathology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Kung-Chao
KC
Chang
Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, No. 35, Xiaodong Rd, Tainan, 70457, Taiwan.
Kuen-Jer
KJ
Tsai
Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
H Sunny
HS
Sun
Division of Infectious Diseases, National Health Research Institutes, Tainan, Taiwan.
Ih-Jen
IJ
Su
Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, No. 35, Xiaodong Rd, Tainan, 70457, Taiwan. pchiang@mail.ncku.edu.tw.
Po-Min
0000-0002-4950-9292
PM
Chiang
D002021:Buffers; D002452:Cell Count; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D008274:Magnesium / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D026723:Polyadenylation / Q000187:drug effects; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription / Q000187:drug effects; D059010:Single-Cell Analysis; D059467:Transcriptome / Q000187:drug effects / Q000235:genetics*
D002021:Buffers; D009711:Nucleotides; D012333:RNA, Messenger; D012313:RNA; D008274:Magnesium
Endothelium; Low cell number; Pluripotent stem cells; Total RNA; Transcriptome sequencing