Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium

Yi Hsuan Lee, Ya Wen Hsueh, Yao Hung Peng, Kung-Chao Chang, Kuen-Jer Tsai, Hsiao-Fang Sun, Ih Jen Su, Po-Min Chiang

Research output: Contribution to journalArticle

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Original languageEnglish
Article number22
JournalBMC Biology
Volume15
Issue number1
DOIs
Publication statusPublished - 2017 Mar 21

Fingerprint

endothelium
Transcriptome
transcriptome
Endothelium
Amplification
RNA
amplification
Cell Count
MicroRNAs
Untranslated RNA
Messenger RNA
messenger RNA
Stem cells
cells
complementary DNA
Complementary DNA
Throughput
microRNA
Purification
Buffers

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Structural Biology
  • Ecology, Evolution, Behavior and Systematics
  • Physiology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Plant Science
  • Developmental Biology
  • Cell Biology

Cite this

@article{a99a613d9bf2488b8d8eb0a57417ab4b,
title = "Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium",
abstract = "Background: 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. Methods: 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. Results: 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. Conclusions: 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.",
author = "Lee, {Yi Hsuan} and Hsueh, {Ya Wen} and Peng, {Yao Hung} and Kung-Chao Chang and Kuen-Jer Tsai and Hsiao-Fang Sun and Su, {Ih Jen} and Po-Min Chiang",
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Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium. / Lee, Yi Hsuan; Hsueh, Ya Wen; Peng, Yao Hung; Chang, Kung-Chao; Tsai, Kuen-Jer; Sun, Hsiao-Fang; Su, Ih Jen; Chiang, Po-Min.

In: BMC Biology, Vol. 15, No. 1, 22, 21.03.2017.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium

AU - Lee, Yi Hsuan

AU - Hsueh, Ya Wen

AU - Peng, Yao Hung

AU - Chang, Kung-Chao

AU - Tsai, Kuen-Jer

AU - Sun, Hsiao-Fang

AU - Su, Ih Jen

AU - Chiang, Po-Min

PY - 2017/3/21

Y1 - 2017/3/21

N2 - Background: 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. Methods: 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. Results: 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. Conclusions: 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.

AB - Background: 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. Methods: 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. Results: 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. Conclusions: 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.

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