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. 2014 Mar 18;86(6):2867-70.
doi: 10.1021/ac500459p. Epub 2014 Mar 4.

Digital Encoding of Cellular mRNAs Enabling Precise and Absolute Gene Expression Measurement by Single-Molecule Counting

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Free PMC article

Digital Encoding of Cellular mRNAs Enabling Precise and Absolute Gene Expression Measurement by Single-Molecule Counting

Glenn K Fu et al. Anal Chem. .
Free PMC article

Abstract

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

Figures

Figure 1
Figure 1
Transcript counting using MI. (A) mRNAs are labeled using barcoded oligo-dT primers. cDNAs of interest are amplified with gene-specific and universal PCR primers (B). Dye labeled products generated by nested PCR are hybridized to a barcode detector and imaged (E) and counted (F) on a custom-built fluorescent CCD instrument. (C) Perspective view of the imaging and illumination optical train, and translation stage. (D) Optical components: achromatic cemented doublet lens (1), emission bandpass filter (2), camera lens (3), CCD (4), plano-convex lenses (5), excitation bandpass filter (6), rectangular aperture (7), aspheric lens (8), LED (9), and barcode detector (10).
Figure 2
Figure 2
MI measurement accuracy. (A) Measured vs input copies of serial dilutions of a synthetic spike-in RNA. (B) GAPDH mRNA measurements in serial dilutions of liver total RNA vs digital PCR. (C) Technical replicate measurements of GAPDH directly in single K562 cell lysates. Absolute counts for each half cell volume are shown. Error bars show 95% measurement confidence intervals.
Figure 3
Figure 3
Gene expression analysis by MI and sequencing. Ten pg (∼1 cell equivalent) of lymphocyte total RNA was barcoded during RT, and 96 genes were amplified by multiplex PCR and sequenced. Numbers of mapped reads (A) or molecule counts (B) are compared with RPKM values from conventional RNA-Seq of 500 ng (∼50 000 cells) of the same sample (for genes >30 RPKM).

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