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Quantification of nascent transcription by bromouridine immunocapture nuclear run-on RT-qPCR

Abstract

Nuclear run-on (NRO) is a method that measures transcriptional activity via the quantification of biochemically labeled nascent RNA molecules derived from nuclear isolates. Widespread use of this technique has been limited because of its technical difficulty relative to steady-state total mRNA analyses. Here we describe a detailed protocol for the quantification of transcriptional activity in human cell cultures. Nuclei are first isolated and NRO transcription is performed in the presence of bromouridine. Labeled nascent transcripts are purified by immunoprecipitation, and transcript levels are determined by reverse-transcription quantitative PCR (RT-qPCR). Data are then analyzed using standard techniques described elsewhere. This method is rapid (the protocol can be completed in 2 d) and cost-effective, exhibits negligible detection of background noise from unlabeled transcripts, requires no radioactive materials and can be performed from as few as 500,000 nuclei. It also takes advantage of the high sensitivity, specificity and dynamic range of RT-qPCR.

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Figure 1: Complete workflow for NRO, bromouridine (BrU) immunocapture and RT-qPCR.
Figure 2: Examples of data generated by NRO-RT-qPCR.
Figure 3: Detection of short-term changes in transcriptional activity by NRO-RT-qPCR.

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Acknowledgements

T.C.R. is supported by a Medical Research Council UK Centenary Early Career Award. This is The Scripps Research Institute manuscript no. 29005.

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Authors

Contributions

T.C.R., J.R.H., M.U.K., M.S.W., P.K.V. and K.V.M. contributed to the protocol and analyzed data. Experimental work was performed by T.C.R. and J.R.H. All authors contributed to the protocol and analyzed data. T.C.R. wrote the initial draft and all authors contributed to the final draft.

Corresponding author

Correspondence to Thomas C Roberts.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Preparation of nuclear extracts.

Successful isolation of intact nuclei from adherent and suspension cultures. P493-6 and HEK293T cells were harvested by gentle lysis and low speed centrifugation. (a) Whole cells and nuclear extracts were visualised by light microscopy. (b) RNA was extracted from each fraction and 28S and 18S ribosomal RNAs visualised by agarose gel electrophoresis. (c) Protein was harvested from both fractions and LMNB1 and GAPDH expression measured by western blot. Scale bars indicate 50 µm.

Supplementary Figure 2 Validation of NRO-RT-qPCR protocol.

P493-6 nuclei were harvested and RNA extracted either immediately (NRO-) or following NRO transcription (NRO+). (a) Nuclear RNA content was determined by NanoDrop spectrophotometry and (b) pre-immunoprecipitation (pre-IP) NRO-RNAs were quantified by RT-qPCR for MYC, ACTB and GAPDH. (c) Nuclear RNA samples were immunoprecipitated using anti-BrdU antibodies and (post-IP) NRO-RNAs quantified by RT-qPCR. (d) P493-6 nuclei were harvested and NRO transcription performed. NRO reactions were terminated by extracting RNA at a series of time points and NRO-RNAs measured by RT-PCR. (e) NRO was performed in the presence and absence of the RNA polymerase II inhibitor α-amanitin (1 µM) and NRO-RNAs quantified by RT-qPCR. (f) Specificity of anti-BrdU immunoprecipitation was confirmed by comparison to a non-specific anti-IgG control antibody. (g) Immunoprecipitation was performed with a range of input nuclear RNA amounts. (h) NRO was performed using a range of different numbers of nuclei and nuclear RNA content was determined by NanoDrop. (i) Labelled NRO-RNAs derived from different numbers of starting nuclei were quantified for MYC, ACTB, GAPDH and GUSB. Values are mean +/- SEM. Cq, quantification cycle. NRO-RNA quantification was performed using the ΔCq method (i.e. not normalised to a reference gene) and the value of one experimental group returned to a value of one for each assay.

Supplementary Figure 3 Assessment of anti-BrdU antibodies.

(a) Details of the three anti-BrdU antibodies (IIB5, Bu20a and ICR1). (b) NRO was performed on P493-6 nuclei. NRO-RNAs were immunoprecipitated in parallel using IIB5, Bu20a or ICR1 antibodies. The levels of MYC, ACTB and GAPDH NRO-RNAs were determined by RT-qPCR. Values are mean + SEM, n=3.

Supplementary Figure 4 RT-qPCR primer binding locations for MYC, ACTB, GAPDH and GUSB.

In silico PCR was used to map RT-qPCR primer sequences (indicated by black brackets) to the human genome (hg19) for (a) MYC, (b) ACTB, (c) GAPDH, and (d) GUSB.

Supplementary Figure 5 Monitoring background signal using exogenous spike-in RNA controls.

(a) Approach for generation of positive and negative spike-in controls. RLuc and FLuc sequences were amplified from the psiCHECK-2 plasmid in separate PCR reactions to generate in vitro transcription (IVT) templates. IVT products were generated using T7 polymerase in the presence or absence of BrUTP. (b) Generation of IVT templates and IVT products were confirmed by agarose and polyacrylamide gel electrophoresis respectively. (c) NRO was performed in the presence or absence of the exogenous spike-in controls. NRO-RNAs were precipitated and quantified by RT-qPCR. Data were compared relative to input samples that were not precipitated. (d) Addition of the exogenous spike-in controls did not significantly affect the levels of NRO-RNAs for MYC, ACTB or GAPDH. Values are mean + SEM, § not detected. NRO-RNA quantification was performed using the ΔCq method (i.e. not normalised to a reference gene) and the value of one experimental group returned to a value of one for each assay.

Supplementary Figure 6 Considerations for NRO-RT-qPCR assay design.

(a) NRO-RNAs are not spliced or polyadenylated. A gene was selected at random (BRPF3) and publically available GRO-seq (GEO accession number: GSM1006729) and RNA-seq data (from the ENCODE Consortium) visualised in the UCSC genome browser. GRO-seq reads are distributed across the gene and extend after the polyadenylation signal, whereas RNA-seq reads predominantly map to annotated exons. (b) Schematic indicating suitable and unsuitable qPCR assay designs for NRO.

Supplementary Figure 7 Negligible amplification in reverse transcriptase minus controls.

The NRO protocol was performed on P493-6 cells and precipitated MYC NRO-RNAs were analysed by RT-qPCR. Reverse transcription was performed in the presence (RT+) and absence (RT-) of reverse transcriptase enzyme. Raw Cq values (a) and melting curve analyses (b,c) are shown. Values are mean +/- SEM. ND, not detected.

Supplementary Figure 8 Validation of reference genes for NRO-RT-qPCR.

Human NRO-RT-qPCR reference gene assays were validated by analyzing 10 fold dilution series of human genomic DNA. Standard curves and melt analysis are shown for each assay.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1, Supplementary Results, Supplementary Discussion and Supplementary Methods (PDF 2058 kb)

Supplementary Data

Additional validation data for NRO-RT-qPCR reference genes. (XLSX 15 kb)

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Roberts, T., Hart, J., Kaikkonen, M. et al. Quantification of nascent transcription by bromouridine immunocapture nuclear run-on RT-qPCR. Nat Protoc 10, 1198–1211 (2015). https://doi.org/10.1038/nprot.2015.076

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