Dec 26, 2025
SELECT-based quantification of site-specific m6A modification
- Xin Xu1,
- Yujuan Wang1
- 1Princess Margaret Cancer Centre
Protocol Citation: Xin Xu, Yujuan Wang 2025. SELECT-based quantification of site-specific m6A modification. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g77rzqgwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 22, 2025
Last Modified: December 26, 2025
Protocol Integer ID: 235607
Keywords: SELECT assay, m6A quantification, site-specific m6A, RNA modification, epitranscriptomics, qPCR, RNA methylation, validation assay, specific quantification of m6a rna modification, m6a rna modification, specific m6a modification, specific m6a modification this protocol, using total rna, total rna, m6a, qpcr amplification, based qpcr amplification, qpcr, detection of select product
Disclaimer
This protocol is provided for research use only and may require optimization depending on experimental conditions.
Abstract
This protocol describes SELECT (single-base elongation- and ligation-based qPCR amplification) assays for site-specific quantification of m6A RNA modification using total RNA, followed by qPCR-based detection of SELECT products.
Materials
- 40 nM Up primer**
- 40 nM Down primer**
- 5 μM dNTPs (NEB, N0447S)
- 1x CutSmart buffer (NEB, B7204S)
- 0.01 U Bst 2.0 DNA polymerase (NEB, M0537S)
- 0.5 U SplintR ligase (NEB, M0375S)
- 10 nmol ATP (NEB, P0756S)
- SYBR Green Master Mix (Applied Biosystems, A25742)
Troubleshooting
Before start
- Use RNase-free tubes, tips, and reagents throughout.
- Prepare all primer working solutions in nuclease-free water.
- Thaw enzymes on ice and keep on ice until use.
- All centrifugation steps are performed briefly to collect liquid unless otherwise specified.
Annealing
Prepare the annealing reaction for each sample by mixing:
500 ng total RNA
40 nM Up primer
40 nM Down primer
5 μM dNTPs (NEB, N0447S)
1x CutSmart buffer (NEB, B7204S)
Adjust volume to 17 μL with nuclease-free water.
Perform primer annealing using the following temperature program:
90°C for 1 min
80°C for 1 min
70°C for 1 min
60°C for 1 min
50°C for 1 min
40°C for 6 min
Elongation and ligation
Prepare anenzyme mixture containing:
0.01 U Bst 2.0 DNA polymerase (NEB, M0537S)
0.5 U SplintR ligase (NEB, M0375S)
10 nmol ATP (NEB, P0756S)
Add 3 μL enzyme mixture to each annealed reaction to reach a final volume of 20 μL.
Incubate reactions using the following program:
40°C for 20 min
80°C for 20 min
Hold at 4°C
qPCR detection
Dilute SELECT reaction products five-fold with nuclease-free water.
Use 2 μL diluted product as template for qPCR amplification using:
SELECT common primers
SYBR Green Master Mix (Applied Biosystems, A25742)
Perform qPCR according to the manufacturer’s instructions.
Data interpretation
The abundance of SELECT qPCR products reflects the relative methylation level at the interrogated adenosine site.
All SELECT assays are performed with at least three independent biological replicates.
Protocol references
Xiao Y, Wang Y, Tang Q, Wei L, Zhang X, Jia G. An Elongation- and Ligation-Based qPCR Amplification Method for the Radiolabeling-Free Detection of Locus-Specific N6 -Methyladenosine Modification. Angew Chem Int Ed Engl. 2018 Dec 3;57(49):15995-16000. doi: 10.1002/anie.201807942. Epub 2018 Nov 8. PMID: 30345651.
Acknowledgements
**Notes**
- Primer sequences used for SELECT assays are provided in Supplementary Table 8_.
- Annealing temperature steps are critical for specificity and should not be shortened.
- Relative comparisons should be performed using matched input RNA amounts across samples.