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International Corporation

BRIC Kit5-Bromouridine

Immunoprecipitation Chase Kit for RNA stability and half-life analysis

• Investigate RNA metabolism in living cells

• Drug target and biomarker discovery

• Lower toxicity

• Easy to use and reproducible

Conventional RNA stability and half-life analysis methods in living cells are difficult to reproduce and often inaccurate. The BRIC Kit is based on pulse chase of BrU and immunoprecipitation that provides researchers with more reproducible and accurate results. Our kit enables researchers to complete their testing in fewer steps, less cytotoxicity and with all the necessary reagents in a convenient, ready-to-use format.

Epigenetics

Assay procedure

Time course cell harvesting IP of BrU-labeled RNAs

BrU

Analysis of BrU labeled RNA• Deep sequencing• RT-qPCR• Microarray

24 h

2412840

Isolation of BrUlabeled RNA

Wash and change medium

Cell seedingPulse label with BrU

RNA extraction Anti-BrdU mAb(Cross-react with BrU)

Total RNA

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RNA stability analysis example

Code No. Product Size

RN1007-RN1008 BRIC Kit for RNA Stability and Half-Life Analysis 20 assays

For research use only, not for use in diagnostic procedures MC-RUO-001

1,800

1,600

1,400

1,200

1,000

800

600

400

200

0

100

90

80

70

60

50

40

30

20

10

0Fold

loss

of l

abel

ed R

NA

in 2

4 ho

urs

18S rR

NA

ACTB

PPIB

TUBA1A1

BNIP3L

TBP

HIF-1α

ADM

18S rR

NA

ACTB

PPIB

TUBA1A1

BNIP3L

TBP

HIF-1α

ADM

Fold

loss

of l

abel

ed R

NA

in 2

4 ho

urs

HeLa

K562

293T

HT29

Jurkat

HEK293

T-47D 0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

18S rR

NA

ACTB PPIB

TUBA1A1

BNIP3L

TBP

HIF-1a

ADM

Fold

loss

of l

abel

ed R

NA

in 2

4 ho

urs

HT29

Jurkat

HEK293

T-47D

Enlarged figure

qRT-PCR analysis for isolated RNA also indicated that a stability of housekeeping genes such as 18S rRNA and ACTB is higher than mRNAs of HIF-1α and ADM.

These results also indicated that labeling efficiencies of BrU in Jurkat, HEK293 and T-47D is lower than other cell lines.

Culture(h)

BrU

BRIC

BrU pulse24 h

2412840

RNA extraction

y = 72.762e-0.019x

0.1

1

10

100

0 5 10 15 20 25Rel

ativ

e R

NA

rem

ain

ing

(%)

Time (hours)

18S rRNA

Housekeeping gene

t1/2=36.5 h

y = 178.59e-0.034x

0.1

1

10

100

0 5 10 15 20 25Rel

ativ

e R

NA

rem

ain

ing

(%)

Time (hours)

ACTB

Housekeeping gene

t1/2=20.4 h

y = 133.44e-0.214x

0.1

1

10

100

0 5 10 15 20 25Rel

ativ

e R

NA

rem

ain

ing

(%)

Time (hours)

HIF -1α

Transcription factor

t1/2=3.2 h

y = 67.725e-0.691x

0.1

1

10

100

0 5 10 15 20 25Rel

ativ

e R

NA

rem

ain

ing

(%)

Time (hours)

ADM

Bioactive peptide

t1/2=1.0 h

qRT-PCR analysis for isolated RNA indicated that a stability of housekeeping genes such as 18S rRNA and ACTB (β-actin) is higher than mRNAs of HIF-1α (Hypoxia-inducible factor-1α) and ADM (Adrenomedullin).

RNA half-life analysis in HeLa cells