Two methods for full-length RNA sequencing for low quantities of cells and single cells Academic Article uri icon

Overview

MeSH Major

  • Gene Expression Profiling
  • High-Throughput Nucleotide Sequencing
  • Nucleic Acid Amplification Techniques
  • RNA, Messenger
  • Single-Cell Analysis

abstract

  • The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3' biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling.

publication date

  • January 8, 2013

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC3545756

Digital Object Identifier (DOI)

  • 10.1073/pnas.1217322109

PubMed ID

  • 23267071

Additional Document Info

start page

  • 594

end page

  • 9

volume

  • 110

number

  • 2