User manual

58 | Droplet Digital
™
PCR Applications Guide
Gene Expression
Similar to RT-qPCR, reverse transcription Droplet Digital
™
PCR (RT-ddPCR) approaches
can also use a one-step or two-step protocol. Choose your approach based on your
experimental purpose.
Two-Step Reverse Transcription ddPCR
Obtain RNA
Use a commercial kit to extract RNA. Store at 100 ng/µl in 1/10 TE buffer (0.1x TE)
or other appropriate buffer at –80°C.
Generate cDNA
Generate cDNA according to standard protocols. We recommend Bio-Rad’s iScript
™
Select cDNA synthesis kit for oligo(dT) or gene-specific priming. For random priming,
we recommend the iScript advanced cDNA synthesis kit for RT-qPCR. Follow the
instructions in the manual of the respective cDNA kit.
Once the reverse transcription is complete, reduce the concentration of cDNA to about
0.2 ng/µl RNA equivalent and use 5 µl per Droplet Digital PCR (ddPCR
™
) reaction (total
volume 20 µl). Typically 1 ng (5 µl of 0.2 ng/µl) of RNA-equivalent cDNA per ddPCR reaction
is adequate to measure most of the transcripts reliably. If the RNA is highly degraded or very
low quality, as is the case with RNA from formalin-fixed, paraffin-embedded (FFPE) samples,
or the transcript of interest is expected to be found at less than 1 copy/cell, such as a
transcript from a cancer cell in a large background of normal cells, the previous guideline
of 1 ng/reaction no longer holds. Under these situations, up to 10 µl of the RT reaction
can be added to one ddPCR reaction without altering its performance.
One-Step RT-ddPCR Kit for Probes
The one-step RT-ddPCR kit for probes follows the same workflow as the ddPCR supermix
for probes, with the benefit that you can now directly partition sample RNA instead of
DNA. The sample is partitioned into 20,000 droplets, with target and background RNA
randomly distributed among the droplets. An RNase inhibitor included in the formulation
minimizes template degradation during reaction setup and droplet generation. After reverse
transcription, the resulting cDNA is amplified for target detection using TaqMan hydrolysis
probes. After PCR amplification, each droplet provides a fluorescent positive or negative
signal indicating the target RNA was present or not present after partitioning. Each droplet
provides an independent digital measurement. Positive and negative droplets are counted
and software calculates the concentration of target RNA as copies/µl.