BRIC Kit is designed to analyze stability/half-life of mRNA and non-coding RNA (ncRNA). This kit is also useful to identify novel ncRNA and transcripts based on their half-lives.
- For characterization of mRNA and ncRNA
- For searcing new ncRNA biomarkers and drug targets
- For discovering new RNA degradation control pathways
Transcriptional inhibitors such as actinomycin D have been used in the analysis of stability and half-life of RNA; however, the use of transcriptional inhibitors alters the stability and localization of RNA, and it has been shown to interfere with the results of analysis.
In 5-bromouridine immunoprecipitation chase (BRIC) method, RNA is transcriptionally pulse-labeled with 5-bromouridine (BrU) and BrU-labeled nascent RNA is isolated using anti-bromodeoxyuridine (BrdU) antibody through its cross-reactivity. This enables determination of RNA stability by chasing chronological decreases of BrU-labeled RNA under physiologically undisturbed conditions.
In combination with RT-qPCR, deep sequencing or microarray, BRIC Kit allows identification of novel ncRNA and transcripts as well as analysis of
In the BRIC protocol, cells are pulse-labeled with BrU for constant period and washed with PBS to remove the BrU-containing medium, and then cells are chronologically harvested, followed by preparation of total RNA including newly BrU-labeled RNA. The BrU-labeled RNA can be specifically immunoprecipitated with Anti-BrdU mAb provided by MBL, followed by isolation of BrU-labeled RNA from immunocomplex on carrier material, such as protein G magnetic beads. The isolated BrU-labeled RNA can be analyzed to determine its own stability and half-life by various methods in molecular biology – RT-qPCR, deep sequencing or microarray.
BRIC Kit provides:
1) BrU for RNA labeling and high-quality Anti-BrdU antibody for isolation of BrU-RNA
2) Protein G-Magnetic beads which reduce background caused by non-specific binding of nucleic acids in RNA-IP
3) Spike-in control for the normalization of RNA-IP results
4) Phenol-free RNA isolation reagents which reduce hazardous waste
The kit offers three different RNA isolation protocols on customer's demand:
Examples of BRIC Analysis
Target RNA decay in 24 hours
HeLa, K562, 293T, HT29, Jurkat, HEK293 and T-47D cells were pulse-labeled with 150 µM BrU for 24 hours. Then, the cells were washed and harvested at chase time 0 and 24 hours. After RNA extraction, BrU-labeled RNA was isolated by BRIC Kit. The isolated BrU-labeled RNAs were analyzed by RT-qPCR. The RNAs derived from housekeeping genes such as 18S rRNA and ACTB were stable in most of cell lines, while HIF-1α and ADM were unstable. The labeling efficiency was low in Jurkat, HEK293 and T-47D cells, which indicates labeling efficiency varies depending on the cell lines.
Measurement of half-life of target RNA by RT-qPCR
HeLa cells were pulse-labeled with 150 µM BrU for 24 hours. Then, cells were washed and harvested at chase time 0, 4, 8, 12 and 24 hours. After RNA extraction, BrU-labeled RNA was isolated by BRIC Kit. Isolated BrU-labeled RNA was analyzed by RT-qPCR.
As expected, the transcripts derived from housekeeping genes, such as 18S rRNA and ACTB, showed relatively long half-lives, while HIF-1α and ADM* showed much shorter half-lives.
*ADM gene encodes a potent hypotensive peptide which plays important roles in both normal and disease conditions.
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