Thermo Fisher PrimeFlow User Manual

Getting started guide

PrimeFlow RNA Assay

Conjugated

antibodies

Flow RNA Flow cytometry

instrument

Contents

1. Introduction 3

2. Workflow summary 4

3. Things to consider 6

4. Checklist—what you’ll need 8

5. FAQs 9

6. Ordering information 10

7. Additional resources 10

1. Introduction

Label probe

What is the PrimeFlow RNA Assay?

The Invitrogen™ PrimeFlow™ RNA Assay employs
fluorescence in situ hybridization (FISH) with branched-DNA
(bDNA) signal amplification for the simultaneous detection
of up to four RNA targets. This assay can also be used
in combination with immunolabeling of both cell-surface
and intracellular proteins using fluorophore-conjugated
antibodies and detection by flow cytometry. The PrimeFlow
RNA Assay can detect messenger RNA (mRNA), long
noncoding RNA (lncRNA), and microRNA (miRNA).

What is bDNA signal amplification?

bDNA signal amplification is achieved through sequential
hybridization steps with preamplifiers, amplifiers, and
fluorophore-conjugated label probes (Figure 1). A fully
assembled signal amplification “tree” has 400 label-probe
binding sites. When all target-specific oligonucleotides in
the probe set bind to the target RNA transcript, 8,000- to
16,000-fold amplification can be achieved.

The purpose of this guide is to provide all the necessary
information to help you get started with the PrimeFlow RNA
Assay and walk you through the design and workflow of an
experiment using the PrimeFlow RNA Assay.

BA

Preamplifier

mRNA

Probe set

C D

Amplifier

Figure 1. Signal amplification by sequential hybridization of oligonucleotides. (A) Gene-specific probe sets are hybridized to target RNA transcripts.
(B) Preamplifier (“trunk”) binds to a probe set. (C) Amplifiers (“branches”) bind to multiple sites on the preamplifier. (D) Fluorophore-conjugated label

probes (“leaves”) bind to multiple sites on the amplifiers.

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