Simultaneously visualize RNA, with single-molecule sensitivity, and protein in single cells

The complex interactions between transcription, translation, and post-translational modifications are hidden from view in typical endpoint assays that measure either RNA or protein levels. While in situ hybridization (ISH) provides a method for examining the levels of specific RNA transcripts in individual cells, and immunocytochemistry (ICC) utilizes antibodies to visualize the localization of specific proteins (and protein modifications), the ability to simultaneously observe RNA and protein in a single cell has been thwarted by the incompatibility of ICC and ISH protocols.

Further complicating this analysis, traditional ISH methods lack sensitivity for detecting low-abundance RNA species and have limited multiplexing capability that further constrain its compatibility with other cell assays.

Invitrogen™ ViewRNA™ Cell Plus Assay is a novel method that combines ICC with the Invitrogen™ ViewRNA™ technology, a proprietary fluorescence ISH (FISH) and sequential branched-DNA (bDNA) amplification technique, to visualize RNA with single-molecule sensitivity along with protein in individual cells. This assay enables simultaneous detection of up to three RNA targets in combination with immunophenotyping for cell surface and intracellular proteins using both indirect and direct ICC, which allows for in-depth characterization of specific cell subpopulations. The combined visualization of RNA and protein expression at the single-cell level is a valuable tool that has broad implications for advancing research across multiple fields of biology.

Technology overview

FISH is a powerful technique that enables the specific detection of target RNA localization in fixed cells. Traditional FISH techniques using large oligonucleotide sequences labeled with one to five fluorophores are generally limited by high background and low sensitivity due to non-specific binding and insufficient signal amplification.

ViewRNA Cell Assays incorporate a proprietary probe set design and bDNA signal amplification technology. A target-specific Probe Set of approximately 20 oligonucleotide pairs hybridizes to the target RNA of interest. Signal amplification is achieved through specific hybridization of adjacent oligonucleotide pairs to bDNA structures, which are formed by Pre-amplifiers, Amplifiers, and fluorochrome-conjugated Label Probes. This methodology results in greater specificity, lower background, and higher signal-to-noise ratios. ViewRNA Cell Assays are the most sensitive and specific RNA ISH methods on the market.

In comparison, to create images with similar discernible spots, traditional FISH techniques can require a 600-times longer exposure and a 100-times greater camera gain than ViewRNA ISH Cell Assays. Furthermore, under equivalent imaging conditions, ViewRNA ISH Cell Assay is 100-times brighter, creating a minimum two- to three-times higher signal-to-noise ratio (Nat Methods 10:1127 (2013) ).

Search for available (in stock) probe sets

Assay kits and supporting products

The ViewRNA Cell Plus Assay contains all the reagents needed to conduct the assay. Positive control probes and target probes for genes of interest are sold separately.

Antibody compatibility

To date, we have evaluated the performance of more than 100 ICC-compatible antibodies (comprised of a mix of unconjugated/purified, biotinylated, and fluorophore-conjugated format; see table below) for use in ViewRNA Cell Plus Assay. To accomplish this, antibody performance was tested either in the context of the full assay (antibody staining and RNA detection) or by using Invitrogen™ ViewRNA™ Cell Plus Fixation/Permeabilization Buffer Set (Cat. No. 00-19001) according to the antibody staining portion of the protocol. We found that 95% of the antibodies tested that perform optimally with formaldehyde-fixed cells also perform well with the ViewRNA Cell Plus Assay. Antibodies that require methanol fixation were generally found to be unsuitable for this assay.

In this evaluation, we have assessed a broad panel of antibodies with specificities against structural proteins, transcription factors, organelles, and surface markers. Our results show that signal intensity and resolution of detail is maintained with ViewRNA Cell Plus Assay compared to our standard immunocytochemistry protocol. Based on our studies, we conclude that antibodies which are suitable for ICC or immunohistochemistry (IHC) (i.e., optimized for formaldehyde fixation) are likely to be compatible with ViewRNA Cell Plus Assay. Nevertheless, we recommend using ViewRNA Cell Plus Fixation/Permeabilization Buffer Set (Cat. No. 00-19001) to establish acceptable performance criteria prior to running the complete ViewRNA Cell Plus Assay.

Species

Specificity

Clone

Cat. No.

Conjugate

Concentration tested

Recommended

Human, Mouse, Rat, Canine, Bovine, Guinea Pig, Pig, Horse

Actin

HHF35

14-6496

Purified

10 µg/mL

Human, Mouse, Rat, Canine, Bovine, Guinea Pig, Pig, Horse

Actin

HHF35

53-6496

Invitrogen™ Alexa Fluor™ 488

30 µg/mL

Human

AFP (alpha fetoprotein)

1E8

14-9499

Purified

5 µg/mL

Human

AHR

FF3399

14-9854

Purified

20 µg/mL

Human

ALDH1L1

7G8

14-9595

Purified

1 µg/mL

Human

alpha-Catenin

1G5

14-9120

Purified

5 µg/mL

Human

Arginase-1

sl6arg

53-9779

Alexa Fluor 488

20 µg/mL

Human

Arginase-1

sl6arg

50-9779

Invitrogen™ eFluor™ 660

5 µg/mL

Human

Arginase-1

sl6arg

14-9779

Purified

5 µg/mL

Human, Mouse

B220

RA3-6B2

53-0452

Alexa Fluor 488

20 µg/mL

Human, Mouse, Rat, Bovine

Beta tubulin class III

2G10-TB3

14-4510

Purified

5 µg/mL

Human, Mouse, Rat, Bovine

Beta tubulin class III

2G10-TB3

53-4510

Alexa Fluor 488

10 µg/mL

Human, Mouse, Rat, Bovine

Beta tubulin class III

2G10-TB3

50-4510

eFluor 660

10 µg/mL

Human, Mouse, Rat, Bovine

Beta tubulin class III

2G10-TB3

41-4510

Invitrogen™ eFluor™ 570

5 µg/mL

Human

Brachyury

X1AO2

14-9770

Purified

5 µg/mL

Mouse

CD3

17A2

50-0032

eFluor 660

10 µg/mL

Human, Mouse and Canine

CD324, E-Cadherin

DECMA-1

53-3249

Alexa Fluor 488

20 µg/mL

Mouse

CD4

RM4-5

13-0042

Biotin

10 µg/mL

Mouse

CD4

RM4-5

41-0042

eFluor 570

5 µg/mL

Mouse

CD8

53-6.7

50-0081

eFluor 660

10 µg/mL

Mouse

CD8a

4SM15

13-0808

Biotin

10 µg/mL

Mouse

CD11b

M1/70

50-0112

eFluor 660

5 µg/mL

Human, Mouse, Rat, Chicken

Connexin 32

2A

14-9759

Purified

5 µg/mL

Human, Mouse, Rat

Connexin 43

1A

50-4759

eFluor 660

10 µg/mL

Human

Cox IV

IB52C31H10

14-9775

Purified

1 µg/mL

Human

Cox IV

IB52C31H10

41-9775

eFluor 570

1 µg/mL

Human

CoxIV

IB52C31H10

53-9775

Alexa Fluor 488

10 µg/mL

Human

Cytokeratin 7

LP5K

14-9005

Purified

10 µg/mL

Human, Mouse, Rat

Early endosome antigen 1

1G11

14-9114

Purified

1 µg/mL

Human

CD326 EpCAM

1B7

14-9326

Purified

10 µg/mL

Human

ER alpha

tbykt

14-9740

Purified

1 µg/mL

Human

Fibronectin

FN-3

14-9869

Purified

10 µg/mL

Human, Mouse, Rat, Pig

GFAP

GA5

53-9892

Alexa Fluor 488

10 µg/mL

Human

Golgin-97

CDF4

53-9767

Alexa Fluor 488

10 µg/mL

Human

Golgin-97

CDF4

50-9767

eFluor 660

5 µg/mL

Human

Golgin-97

CDF4

14-9767

Purified

1 µg/mL

Human, Mouse, Rat

Grim-19

1A8

14-9937

Purified

5 µg/mL

Human

Her2

MJD2

14-9757

Purified

5 µg/mL

Human

Her2

MJD2

41-9757

eFluor 570

5 µg/mL

Human, Mouse

Her4

HFR1

14-9687

Purified

10 µg/mL

Human

Ki-67

20Raj1

42-5699

Invitrogen™ eFluor™ 615

1 µg/mL

Human

Ki-67

20Raj1

50-5699

eFluor 660

20 µg/mL

Human

Ki-67

20Raj1

11-5699

FITC

10 µg/mL

Human

Ki-67

20Raj1

14-5699

Purified

1 µg/mL

Human

Ki-67

20Raj1

13-5699

Biotin

5 µg/mL

Human

Ki-67

20Raj1

41-5699

eFluor 570

5 µg/mL

Mouse, Rat

Ki-67

SolA15

41-5698

eFluor 570

0.5 µg/mL

Mouse, Rat

Ki-67

SolA15

50-5698

eFluor 660

10 µg/mL

Hu, m, Rt

MAP2

AP20

41-9763

eFluor 570

5 µg/mL

Mouse, Rat

MASH1

24B72D11

14-5794

Purified

20 µg/mL

Human, Mouse, Rat, Dog

Myogenin

F5D

53-5643

Alexa Fluor 488

5 µg/mL

Human, Mouse, Rat, Dog

Myogenin

F5D

14-5643

Purified

1 µg/mL

Human, Mouse, Rat, etc.

Myosin

MF20

53-6503

Alexa Fluor 488

5 µg/mL

Human, Mouse, Rat, etc.

Myosin

MF20

50-6503

eFluor 660

10 µg/mL

Mouse

Nanog

eBioMLC-51

50-5761

eFluor 660

10 µg/mL

Mouse

Nanog

eBioMLC-51

53-5761

Alexa Fluor 488

10 µg/mL

Human

Nestin

10C2

53-9843

Alexa Fluor 488

10 µg/mL

Human

Nestin

10C2

14-9843

Purified

5 µg/mL

Human, Mouse

OCT3/4

EM92

41-5841

eFluor 570

5 µg/mL

Human, Mouse, Rat

PCNA

PC10

14-9910

Purified

1 µg/mL

Human, Mouse, Rat

Phospho-ATM

10H11.E12

14-9046

Purified

10 µg/mL

Human, Mouse

Phospho-ERK1/2

MILAN8R

14-9109

Purified

5 µg/mL

Human, Mouse

Phospho-H2AX

CR55T33

14-9865

Purified

5 µg/mL

Human

Progesterone receptor

KMC912

13-9764

Biotin

2.5 µg/mL

Human

Progesterone receptor

KMC912

14-9764

Purified

1 µg/mL

Human

Progesterone receptor

KMC912

50-9764

eFluor 660

20 µg/mL

Human

Snail1

20C8

53-9859

Alexa Fluor 488

20 µg/mL

Human

Snail1

20C8

14-9859

Purified

5 µg/mL

Human, Mouse

Sox2

Btjce

41-9811

eFluor 570

5 µg/mL

Human, Mouse

Sox2

Btjce

50-9811

eFluor 660

10 µg/mL

Human, Mouse

Sox2

Btjce

14-9811

Purified

5 µg/mL

Human, Mouse

Sox9

GMPR9

53-9765

Alexa Fluor 488

10 µg/mL

Human, Mouse

Sox9

GMPR9

14-9765

Purified

1 µg/mL

Human

Survivin

STLALYV

53-9176

Alexa Fluor 488

10 µg/mL

Human, Mouse, Rat

Thrombospondin-1

A6.1

14-9756

Purified

1 µg/mL

Human, Mouse, Rat, etc.

Tubulin

DM1A

42-4502

eFluor 615

1 µg/mL

Human, Mouse, Rat, etc.

Tubulin

DM1A

53-4502

Alexa Fluor 488

5 µg/mL

Human, Mouse, Rat, etc.

Tubulin

DM1A

14-4502

Purified

5 µg/mL

Human, Mouse, Rat, etc.

Tubulin

DM1A

13-4502

Biotin

10 µg/mL

Human, Rat, Chicken, Canine

Vimentin

V9

41-9897

eFluor 570

1 µg/mL

Human, Rat, Chicken, Canine

Vimentin

V9

50-9897

eFluor 660

5 µg/mL

Human, Mouse, Rat, Avian

Vinculin

7F9

41-9777

eFluor 570

10 µg/mL

Human, Mouse, Rat, Avian

Vinculin

7F9

53-9777

Alexa Fluor 488

20 µg/mL

Human, Mouse, Rat, Avian

Vinculin

7F9

14-9777

Purified

5 µg/mL

Mouse, Rat, Canine

ZO-1

R26.4C

41-9776

eFluor 570

1 µg/mL

Human

CD3

OKT3

50-0037

eFluor 660

0.12 µg/ml

Human

CD4

N1UG0

41-2444

eFluor 570

20 µg/mL

Mouse

FoxP3

FJK-16s

53-5773

Alexa Fluor 488

10 µg/mL

Not recommended 

Human, Mouse

B220

RA3-6B2

50-0452

eFluor 660

10 µg/mL

Mouse

CD4

GK1.5

53-0041

Alexa Fluor 488

10 µg/mL

Mouse

CD4

GK1.5

50-0041

eFluor 660

5 µg/mL

Mouse, Rat

Nestin

Rat-401

14-5843

Purified

10 µg/mL

Mouse, Human, Rat, etc.

Cytokeratin

AE1/AE3

42-9003

eFluor 615

5 µg/mL

Mouse, Human, Rat, etc.

Cytokeratin

AE1/AE3

41-9003

eFluor 570

1 µg/mL

Mouse, Human, Rat, etc.

Cytokeratin

AE1/AE3

53-9003

Alexa Fluor 488

1 µg/mL

Human, Mouse, Rat, Chicken

Connexin 32

2A

53-9759

Alexa Fluor 488

1 µg/mL

Human, Mouse, Rat

Connexin 43

1A

14-4759

Purified

5 µg/mL

Human

Heat shock protein 27

STRSN

14-9112

Purified

1 µg/mL

Mouse

MHC Class II

M5/114.15.2

11-5321

FITC

20 µg/mL

Human, Mouse, Rat

PCNA

PC10

42-9910

eFluor 615

0.5 µg/mL

Human

CD8

AMC908

50-0008

eFluor 660

5 µg/mL

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