Abstract
This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named 'Worm-to-CT', allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.
Highlights
The optimization of single-cell RNA sequencing and qPCR revealed that transcriptional pulses or bursts can lead to massive variation in the number of RNA molecules per cell[1]
The optimization of high-throughput multiplexed qPCR technologies has proven useful for mammalian single-cell studies, in particular when studying the expression of rare transcripts[14,15]
The results were comparable between both methods and comparable to previous reports[23]. These results indicate that the Worm-to-CT method is a valid alternative to standard complementary DNA (cDNA) synthesis techniques
Summary
The optimization of single-cell RNA sequencing and qPCR revealed that transcriptional pulses or bursts can lead to massive variation in the number of RNA molecules per cell[1]. The optimization of high-throughput multiplexed qPCR technologies has proven useful for mammalian single-cell studies, in particular when studying the expression of rare transcripts[14,15]. This article presents the protocol for rapid cDNA preparation and high-throughput qPCR for both single-worm and batch worm samples; the algorithm will be published elsewhere For this protocol, the organization of each chip should be prepared prior to the experiment. Part II of this protocol (sections 4−13) describes running high-throughput RT-qPCR using nanofluidics, adapted from a protocol developed by Fluidigm[16] This protocol applies to the use of the two types of nanofluidic chips defined earlier, the single-array chip, which can monitor 96 targets into 96 samples (9,216 RT-qPCR reactions total), or the multi-array chip, which functions as subunits of 12 target x 12 samples. For further information regarding any of the materials used in this protocol, refer to the Table of Materials
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