RNA/cRNA Labeling

Fluorophores and haptens, 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).

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

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
pCp-488 NU-1706-488 ✔2
yellow pCp-Cy3 NU-1706-Cy3 ✔2
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

yPAPYeast 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

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.

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.

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

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

yPAPYeast 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

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

yPAPYeast 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

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.

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

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

yPAPYeast 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)
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

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

yPAPYeast 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

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