RNA/cRNA Labeling

Fluorophores and haptens, the latter meaning Biotin, Desthiobiotin, Digoxigenin and Dinitrophenol, are the most commonly used labels for the generation of non-radioactive RNA/cRNA probes. They are enzymatically introduced into RNA/cRNA via modified nucleotides that are incorporated as substitutes for their natural counterparts in either a one-step procedure (introduction of a fluorescent or hapten-modified nucleotide) or a two-step procedure (introduction of a reactive group carrying nucleotide and subsequent fluorescent or hapten labeling).

Label	Labeling Kits	Single Nucleotides
Fluorophore	Fluorescent in vitro Transcription Kits	Fluorescent Nucleotides
Hapten	Biotin&Digoxigenin in vitro Transcription Kits	Biotinylated Nucleotides Digoxigenin-/DNP-modified Nucleotides
Amine		Amine-modified Nucleotides
CLICK	Copper-free CLICk T7 in vitro Transcription Labeling Kits	CLICK-functionalized Nucleotides
Nucleotide dependent

Non-radioactive Labeling of DNA and RNA

Hapten-modified Nucleotides for DNA/RNA Labeling

Fluorescent Nucleotides: A powerful toolbox for labeling of biological macromolecules

Table 1: Standard enzymatic procedures for the preparation of non-radioactive RNA/cRNA probes.

Template	Method	Labeled Probe	Labeling Site	Enzyme
DNA	in vitro Transcription	cRNA	random	T7- / SP6- / T3-RNA Polymerase
RNA	3’ End Labeling	RNA	3’ OH	Terminal deoxynucleotidyl Transferase (TdT)
		RNA	3’ OH	Yeast Poly A Polymerase
		RNA	3’ OH	T4 RNA Ligase
	5’ End Labeling	RNA	5’ OH	T4 Polynucleotide Kinase (T4 PNK)

Fluorescent RNA/cRNA Labeling

Fluorescent Nucleotides

Table 1: Enyzmatic incorporation of fluorescent Nucleotides.

			cRNA Labeling by in vitro Transcription with…			3′-RNA Labeling by…
Emission colour	Nucleotide	Cat. No.	T7 RNA Polymerase	T3 RNA Polymerase	SP6 RNA Polymerase	yPAP	thT	T4 RNA Ligase
UTP
blue	UTP-PEG5-AF405	NU-821-PEG5-AF405	1	n/a	n/a	n/a	n/a	–
green	Fluorescein-12-UTP	NU-821-FAMX	1	n/a	n/a	n/a	n/a	–
	UTP-PEG5-AF488	NU-821-PEG5-AF488	1	n/a	n/a	n/a	n/a	–
	UTP-ATTO-488	NU-821-488	1	n/a	n/a	n/a	n/a	–
yellow-green	UTP-ATTO-532	NU-821-532	1	n/a	n/a	n/a	n/a	–
yellow	UTP-Cy3	NU-821-Cy3	2	n/a	n/a	n/a	n/a	–
	UTP-X-Cy3	NU-821-X-CY3	2	n/a	n/a	n/a	n/a	–
	UTP-ATTO-550	NU-821-550	1	n/a	n/a	n/a	n/a	–
	UTP-PEG5-AF555	NU-821-PEG5-AF555	1	n/a	n/a	n/a	n/a	–
orange	UTP-PEG5-AF594	NU-821-PEG5-AF594	1	n/a	n/a	n/a	n/a	–
red	UTP-Cy5	NU-821-Cy5	2	n/a	n/a	n/a	n/a	–
	UTP-X-Cy5	NU-821-X-CY5	n/a	n/a	n/a	n/a	n/a	–
	UTP-PEG5-AF647	NU-821-PEG5-AF647	1	n/a	n/a	n/a	n/a	–
	UTP-ATTO-647N	NU-821-647N	1	n/a	n/a	n/a	n/a	–
near-IR	UTP-680	NU-821-680	1,2	n/a	n/a	n/a	n/a	–
CTP
yellow	CTP-Cy3	NU-831-Cy3	2	n/a	n/a	n/a	n/a	–
red	CTP-Cy5	NU-831-Cy5	2	n/a	n/a	n/a	n/a	–
Cytidine-5’/3’-phosphate
green	pCp-AF488	NU-1706-AF488	–	–	–	–	–	2
green	pCp-488	NU-1706-488	–	–	–	–	–	2
yellow	pCp-Cy3	NU-1706-Cy3	–	–	–	–	–	2
yellow	pCp-AF555	NU-1706-AF555	–	–	–	–	–	2
orange	pCp-AF594	NU-1706-AF594	–	–	–	–	–	2
red	pCp-Cy5	NU-1706-Cy5	–	–	–	–	–	2
	pCp-643	NU-1706-643	–	–	–	–	–	2
	pCp-AF647	NU-1706-AF647	–	–	–	–	–	2

yPAP: Yeast Poly A Polymerase
TdT: Terminal deoxynucleotidyl Transferase.
¹: in-house tested
²: external reference (see datasheet)
n/a: no data available
– : not applicable

Products & Ordering
Aminoallyl-UTP-PEG5-AF405 NU-821-PEG5-AF405	Fluorescein-12-UTP NU-821-FAMX Fluorescein-X-(5-aminoallyl)-UTP	Aminoallyl-UTP-PEG5-AF488 NU-821-PEG5-AF488
Aminoallyl-UTP-Cy5 NU-821-CY5	pCp-ATTO-488 NU-1706-488	Aminoallyl-UTP-ATTO-488 NU-821-488
Aminoallyl-UTP-X-Cy5 NU-821-X-CY5	pCp-Cy3 NU-1706-CY3	Aminoallyl-UTP-ATTO-532 NU-821-532
Aminoallyl-UTP-PEG5-AF647 NU-821-PEG5-AF647	pCp-AF555 NU-1706-AF555	Aminoallyl-UTP-Cy3 NU-821-CY3
Aminoallyl-UTP-ATTO-647N NU-821-647N	pCp-AF594 NU-1706-AF594	Aminoallyl-UTP-X-Cy3 NU-821-X-CY3
Aminoallyl-UTP-ATTO-680 NU-821-680	pCp-ATTO-643 NU-1706-643	Aminoallyl-UTP-ATTO-550 NU-821-550
5-Propargylamino-CTP-Cy3 NU-831-CY3	pCp-AF647 NU-1706-AF647	Aminoallyl-UTP-PEG5-AF555 NU-821-PEG5-AF555
5-Propargylamino-CTP-Cy5 NU-831-CY5	pCp-Cy5 NU-1706-CY5	Aminoallyl-UTP-PEG5-AF594 NU-821-PEG5-AF594
pCp-AF488 NU-1706-AF488

Fluorescent in vitro Transcription Labeling Kits

Random labeled single-stranded RNA probes can be synthesized by in vitro transcription. The reaction is catalyzed by bacteriophage T7 RNA polymerase that incorporates labeled NTPs (mostly UTP) as substitute for their natural counterpart using linear, RNA probe-encoding DNA as template.

Fluorophore Selection Guide for DNA & RNA Labeling Kits

Products & Ordering
HighYield T7 Fluorescein RNA Labeling Kit RNT-101-FAMX Preparation of randomly Fluorescein-modified RNA probes by in vitro transcription with Fluorescein-12-UTP	HighYield T7 Cy3 RNA Labeling Kit RNT-101-CY3 Preparation of randomly Cy3-modified RNA probes by in vitro transcription with UTP-X-CY3
HighYield T7 Cy5 RNA Labeling Kit RNT-101-CY5 Preparation of randomly Cy5-modified RNA probes by in vitro transcription with UTP-X-CY5	HighYield T7 Atto488 RNA Labeling Kit RNT-101-488 Preparation of randomly Atto488-modified RNA probes by in vitro transcription with UTP-ATTO-488
HighYield T7 AF405 RNA Labeling Kit RNT-101-AF405 Preparation of randomly AF488-modified RNA probes by in vitro transcription with UTP-PEG5-AF405	HighYield T7 AF488 RNA Labeling Kit RNT-101-AF488 Preparation of randomly AF488-modified RNA probes by in vitro transcription with UTP-PEG5-AF488
HighYield T7 AF555 RNA Labeling Kit RNT-101-AF555 Preparation of randomly AF555-modified RNA probes by in vitro transcription with UTP-PEG5-AF555	HighYield T7 AF594 RNA Labeling Kit RNT-101-AF594 Preparation of randomly AF594-modified RNA probes by in vitro transcription with UTP-PEG5-AF594
HighYield T7 AF647 RNA Labeling Kit RNT-101-AF647 Preparation of randomly AF647-modified RNA probes by in vitro transcription with UTP-PEG5-AF647

Hapten Labeling of RNA/cRNA

Biotinylated Nucleotides

Table 1: Enzymatic incorporation of Biotinylated Nucleotides

cRNA Labeling by in vitro Transcription with…					3’-RNA Labeling by…		5’-RNA Labeling by in vitro Transcription with…
Nucleotide	Cat. No.	T7 RNA Polymerase	T3 RNA Polymerase	SP6 RNA Polymerase	PAP	T4 RNA Ligase	T7 RNA Polymerase
UTP
Biotin-11-UTP	NU-821-BIOX	2	2	2	n/a	–	–
Biotin-16-UTP	NU-821-BIO16	2	2	2	n/a	–	–
CTP
Biotin-11-CTP	NU-831-BIOX	2	2	n/a	n/a	–	–
ATP
Biotin-7-ATP N6 position	NU-805-BIO	n/a	n/a	n/a	2	–	–
Biotin-11-ATP 7-Deaza position	NU-957-BIOX	2	n/a	n/a	n/a	–	–
GTP
Biotin-11-GTP	NU-971-BIOX	2	n/a	n/a	n/a	–	–
AMP
Biotin-AMP	NU-894-BIO	–	–	–	–	–	2
Nucleotide-5’/3’-phosphate
pCp-Biotin	NU-1706-BIO	–	–	–	–	2	–
pCp-Desthiobiotin	NU-1706-Desthiobio	–	–	–	–	2	–
5’-Biotin-ApG	NU-888-BIO	–	–	–	–	–	2

yPAP: Yeast Poly A Polymerase
TdT: Terminal deoxynucleotidyl Transferase.
¹: in-house tested
²: external reference (see datasheet)
n/a: no data available
– : not applicable

Products & Ordering
Biotin-11-UTP NU-821-BIOX Biotin-X-(5-aminoallyl)-UTP	Biotin-11-UTP – high concentration NU-821-BIOX-HC Biotin-X-(5-aminoallyl)-UTP	Biotin-16-UTP NU-821-BIO16 Biotin-16-(5-aminoallyl)-UTP
Biotin-11-CTP NU-831-BIOX Biotin-X-5-Propargylamino-CTP	Biotin-11-GTP NU-971-BIOX	pCp-Biotin NU-1706-BIO
N6-(6-Aminohexyl)-ATP-Biotin NU-805-BIO	Biotin-AMP NU-894-BIO	pCp-Desthiobiotin NU-1706-Desthiobio
Biotin-11-ATP NU-957-BIOX	Biotin-16-dUTP NU-803-BIO16 Biotin-16-(5-aminoallyl)-dUTP	5′-Biotin-ApG NU-888-BIO

Digoxigenin-modified Nucleotides

Table 1: Enzymatic incorporation of Digoxigenin-modified Nucleotides

*cRNA Labeling by in vitro* Transcription with…**					3′-RNA Labeling by…
Nucleotide	Cat. No.	T7 RNA Polymerase	T3 RNA Polymerase	SP6 RNA Polymerase	yPAP	TdT	T4 RNA Ligase
UTP
Digoxigenin-11-UTP	NU-821-DIGX	2	2	2	2	n/a	–
(d)/(dd)UTP
Digoxigenin-11-dUTP	NU-803-DIGX	–	–	–	–	2	–
Digoxigenin-11-ddUTP	NU-1619-DIGX	–	–	–	–	2	–

yPAP: Yeast Poly A Polymerase
TdT: Terminal deoxynucleotidyl Transferase.
¹: in-house tested
²: external reference (see datasheet)
n/a: no data available
– : not applicable

Products & Ordering
DIG-11-UTP NU-821-DIGX	DIG-11-dUTP NU-803-DIGX	DIG-11-ddUTP NU-1619-DIGX

Hapten in vitro Transcription Labeling Kits

The optimal ratio of labeled NTP/NTP in terms of product yield and labeling efficiency depends on both the type of labeled NTP and final application. Individual optimization of labeled NTP/NTP ratio can easily be achieved with the single nucleotide format of our HighYield T7 RNA Labeling Kits. They offer maximum optimization flexibility in contrast to fixed NTP labeling mixes combined with high product yields. The combination of (poly)-HRP-conjugated Biotin/Digoxigenin detection reagents with AF 488 (Alexa Fluor® 488)-, AF546 (Alexa Fluor® 546)- or AF594 (Alexa Fluor® 594)-labeled tyramide further increases detection sensitivity of up to 100-fold.

Products & Ordering
HighYield T7 Digoxigenin RNA Labeling Kit RNT-101-DIGX Preparation of randomly Digoxigenin-modified RNA probes by in vitro transcription with Dioxigenin-11-UTP	HighYield T7 Biotin16 RNA Labeling Kit RNT-101-BIO16 Preparation of randomly Biotin-modified RNA probes by in vitro transcription with Biotin-16-UTP
HighYield T7 Biotin11 RNA Labeling Kit RNT-101-BIOX Preparation of randomly Biotin-modified RNA probes by in vitro transcription with Biotin-11-UTP	AF488 tyramide reagent RNT-012 also known as Alexa Fluor® 488 tyramide
AF546 tyramide reagent RNT-013 also known as Alexa Fluor® 546 tyramide	AF594 tyramide reagent RNT-014 also known as Alexa Fluor® 594 tyramide
AF488 Streptavidin Conjugate RNT-015 Streptavidin, Alexa Fluor® 488 Conjugate	AF594 Streptavidin Conjugate RNT-016 Streptavidin, Alexa Fluor® 594 Conjugate

Click Chemistry-based RNA/cRNA Labeling

CLICK-functionalized Nucleotides

Table 1: Enzymatic incorporation of CLICK-functionalized nucleotides.

*cRNA Labeling by in vitro* Transcription with…**					3’-RNA Labeling by…
Nucleotide	Cat. No.	T7 RNA Polymerase	T3 RNA Polymerase	SP6 RNA Polymerase	yPAP	TdT	T4 RNA Ligase
UTP
5-Azidomethyl-UTP	CLK-083	1	n/a	n/a	n/a	n/a	n/a
5-Azido-C3-UTP	NU-157	1,2	n/a	n/a	–	–	–
5-Azido-PEG4-UTP	CLK-054	1	n/a	n/a	n/a	n/a	n/a
5-Ethynyl-UTP	CLK-T08	1	n/a	n/a	–	–	–
DBCO-PEG4-UTP	CLK-055	1	n/a	n/a	n/a	n/a	n/a
Vinyl-UTP	CLK-069	1	n/a	n/a	n/a	n/a	n/a
ATP
8-Azido-ATP	NU-155	–	–	–	2	–	–
3′-Azido-2’,3′-ddATP	NU-882	–	–	–	2	–	–
CTP
5-Azido-PEG4-CTP	CLK-053	1	n/a	n/a	n/a	n/a	n/a
5-DBCO-PEG4-CTP	CLK-056	1	n/a	n/a	n/a	n/a	n/a
3′-dATP (Cordycepin derivative)
N6-Azidohexyl-3′-dATP	NU-1707	–	–	–	n/a	–	–
Cytidine-5’/3′-phosphate
pCp-Azide	NU-1708	–	–	–	–	–	2
CpG
5-DBCO-PEG4-dCpG	CLK-062	n/a	n/a	n/a	n/a	n/a	n/a

yPAP: Yeast Poly A Polymerase
TdT: Terminal deoxynucleotidyl Transferase.
¹: in-house tested
²: external reference (see datasheet)
n/a: no data available
– : not applicable

Products & Ordering
5-Azidomethyl-UTP CLK-083	8-Azido-ATP NU-155
5-Azido-C3-UTP NU-157	3′-Azido-2’,3′-ddATP NU-882
5-Azido-PEG4-UTP CLK-054	5-Azido-PEG4-CTP CLK-053
5-Ethynyl-UTP (5-EUTP) CLK-T08	5-DBCO-PEG4-CTP CLK-056
5-DBCO-PEG4-UTP CLK-055	pCp-Azide NU-1708
5-Vinyl-UTP CLK-069	5-DBCO-PEG4-dCpG CLK-062
N6-(6-Azido)hexyl-3′-dATP NU-1707 (Cordycepin triphosphate derivative)

CLICK T7 in vitro Transcription Labeling Kits

Products & Ordering

HighYield T7 Azide RNA Labeling Kit RNT-101-AZ Preparation of randomly Azide-modified RNA probes by in vitro transcription with 5-Azido-C3-UTP

Selected References

[1] Miller et al. (2018) Near-infrared fluorescent northern blot. RNA 24(12):1871.

Amine Labeling of RNA/cRNA

Amine-modified Nucleotides

Table 1: Enzymatic incorporation of Amine-modified Nucleotides.

*cRNA Labeling by in vitro* Transcription with…**					3’-RNA Labeling by…
Nucleotide	Cat. No.	SP6 RNA Polymerase	T7 RNA Polymerase	T3 RNA Polymerase	yPAP	TdT	T4 RNA Ligase
UTP
5-Aminoallyl-UTP	NU-821	2	2	2	–	n/a	–
CTP
5-Propargylamino-CTP	NU-831	n/a	n/a	n/a	–	n/a	–
ATP
N6-Aminohexyl-ATP	NU-805	n/a	n/a	n/a	–	n/a	–
Cytidine-5’/3′-phosphate
pCp-Amine	NU-1706	–	–	–	–	–	n/a

yPAP: Yeast Poly A Polymerase
TdT: Terminal deoxynucleotidyl Transferase.
¹: in-house tested
²: external reference (see datasheet)
n/a: no data available
– : not applicable

Products & Ordering
Aminoallyl-UTP – Solution NU-821	5-Propargylamino-CTP NU-831	pCp-Amine NU-1706
Aminoallyl-UTP – Solid NU-821-LYO	N6-(6-Aminohexyl)-ATP NU-805

RNA/cRNA Labeling

Table 1: Standard enzymatic procedures for the preparation of non-radioactive RNA/cRNA probes.

Fluorescent RNA/cRNA Labeling

Fluorescent Nucleotides

Table 1: Enyzmatic incorporation of fluorescent Nucleotides.

Fluorescent in vitro Transcription Labeling Kits

Hapten Labeling of RNA/cRNA

Biotinylated Nucleotides

Table 1: Enzymatic incorporation of Biotinylated Nucleotides

Digoxigenin-modified Nucleotides

Table 1: Enzymatic incorporation of Digoxigenin-modified Nucleotides

Hapten in vitro Transcription Labeling Kits

Click Chemistry-based RNA/cRNA Labeling

CLICK-functionalized Nucleotides

Table 1: Enzymatic incorporation of CLICK-functionalized nucleotides.

CLICK T7 in vitro Transcription Labeling Kits

Selected References

Amine Labeling of RNA/cRNA

Amine-modified Nucleotides

Table 1: Enzymatic incorporation of Amine-modified Nucleotides.

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RNA/cRNA Labeling

Table 1: Standard enzymatic procedures for the preparation of non-radioactive RNA/cRNA probes.

Fluorescent RNA/cRNA Labeling

Table 1: Enyzmatic incorporation of fluorescent Nucleotides.

Hapten Labeling of RNA/cRNA

Table 1: Enzymatic incorporation of Biotinylated Nucleotides

Table 1: Enzymatic incorporation of Digoxigenin-modified Nucleotides

Click Chemistry-based RNA/cRNA Labeling

Table 1: Enzymatic incorporation of CLICK-functionalized nucleotides.

Selected References

Amine Labeling of RNA/cRNA

Table 1: Enzymatic incorporation of Amine-modified Nucleotides.

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