Accurate quantification of 3′-terminal 2′-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR

doi:10.1007/s11684-021-0909-7

Front. Med.

2022, Vol. 16

Issue (2) : 240-250 https://doi.org/10.1007/s11684-021-0909-7

RESEARCH ARTICLE

Accurate quantification of 3′-terminal 2′-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR

Yan Kong¹, Huanhuan Hu¹, Yangyang Shan¹, Zhen Zhou¹, Ke Zen¹, Yulu Sun³, Rong Yang²(

), Zheng Fu¹(

), Xi Chen^1,⁴(

)

¹. Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), Institute of Artificial Intelligence Biomedicine, School of Life Sciences, Nanjing University, Nanjing 210023, China
². Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, China
³. Department of General Surgery, Drum Tower Hospital, Medical school of Nanjing University, Nanjing 210008, China
⁴. Research Unit of Extracellular RNA, Chinese Academy of Medical Sciences, Nanjing 210023, China

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Abstract

The continuing discoveries of novel classes of RNA modifications in various organisms have raised the need for improving sensitive, convenient, and reliable methods for quantifying RNA modifications. In particular, a subset of small RNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), are modified at their 3′-terminal nucleotides via 2′-O-methylation. However, quantifying the levels of these small RNAs is difficult because 2′-O-methylation at the RNA 3′-terminus inhibits the activity of polyadenylate polymerase and T4 RNA ligase. These two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification assays. In this study, we profiled 3′-terminal 2′-O-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation treatment. We further compared the efficiency of stem-loop and poly(A)-tailed RT-qPCR in quantifying plant miRNAs in animal tissues and identified stem-loop RT-qPCR as the only suitable approach. Likewise, stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3′-terminal 2′-O-methyl piRNAs in human seminal plasma. In summary, this study established a standard procedure for quantifying the levels of 3′-terminal 2′-O-methyl miRNAs in plants and piRNAs. Accurate measurement of the 3′-terminal 2′-O-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.

Keywords small RNAs 2′-O-methylation sequencing RT-qPCR

Corresponding Author(s): Rong Yang,Zheng Fu,Xi Chen

About author:

Mingsheng Sun and Mingxiao Yang contributed equally to this work.

Online First Date: 11 April 2022 Issue Date: 26 April 2022

Cite this article:

Yan Kong,Huanhuan Hu,Yangyang Shan, et al. Accurate quantification of 3′-terminal 2′-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR[J]. Front. Med., 2022, 16(2): 240-250.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-021-0909-7
https://academic.hep.com.cn/fmd/EN/Y2022/V16/I2/240

Fig.1 Plant miRNAs in the liver of rice-fed mice are detected by oxidative deep sequencing. (A) The levels (total sequencing reads) of animal miRNAs in the livers of rice-fed mice that were unoxidized or oxidized for 40 or 90 min. For normalization, the total sequencing reads of animal miRNAs were normalized to the clean reads of each sample. (B) The levels (total sequencing reads) of plant miRNAs in the livers of rice-fed mice that were unoxidized or oxidized for 40 or 90 min. For normalization, the total sequencing reads of plant miRNAs were normalized to the clean reads of each sample. (C) Relative fold changes of plant and animal miRNAs that were subjected to 40 min of oxidation in comparison with those without oxidation. The sequencing reads of each miRNA were normalized to the clean reads of each sample. (D) Relative fold changes of plant and animal miRNAs that were subjected to 90 min of oxidation in comparison with those without oxidation. The sequencing reads of each miRNA were normalized to the clean reads of each sample.

Fig.2 Comparison of the differences between stem-loop and poly(A)-tailed RT-qPCR in quantifying synthetic 2′-O-methyl plant miRNAs. (A) Schematic diagram of the working model of stem-loop and poly(A)-tailed RT-qPCR. The blue line represents the template miRNA. The black structure or line represents the reverse transcription primer. The red line represents the reverse transcription product. The green line represents the forward primers or reverse primers. “Me” represents the methylation site. (B−F) The absolute levels of 5 synthetic 2′-O-methyl and 2′-OH plant miRNAs (miR156a, miR158a, miR159a, miR166a, and miR168a) detected by stem-loop and poly(A)-tailed RT-qPCR. (G−I) The absolute levels of 3 synthetic 2′-OH animal miRNAs (miR-16, miR-21, and miR-122) detected by stem-loop and poly(A)-tailed RT-qPCR. Data are represented as the mean ± SEM. ***P < 0.001.

Fig.3 Comparison of the differences between stem-loop and poly(A)-tailed RT-qPCR in quantifying 2′-O-methyl plant miRNAs in rice-fed animals. (A and B) The absolute levels of plant miRNAs (e.g., miR156a and miR168a) determined by stem-loop and poly(A)-tailed RT-qPCR in the serum of rice-fed mice. (C−E) The absolute levels of animal miRNAs (e.g., miR-16, miR-21, and miR-122) determined by stem-loop and poly(A)-tailed RT-qPCR in the serum of rice-fed mice. (F and G) The absolute levels of plant miRNAs (miR156a and miR168a) determined by stem-loop and poly(A)-tailed RT-qPCR in the livers of rice-fed mice. miRNA levels were normalized to the total amount of RNA. (H−J) The absolute levels of animal miRNAs (e.g., miR-16, miR-21, and miR-122) determined by stem-loop and poly(A)-tailed RT-qPCR in the livers of rice-fed mice. miRNA levels were normalized to the total amount of RNA. Data are represented as the mean ± SEM (n = 5). **P < 0.01, *** P < 0.001.

Fig.4 Comparison of the differences between stem-loop and poly(A)-tailed RT-qPCR in quantifying synthetic 2′-O-methyl piRNAs and piRNAs in seminal plasma. (A−E) The absolute levels of 5 synthetic 2′-O-methyl and 2′-OH piRNAs (e.g., piR-30198, piR-31068, piR-31925, piR-43771, and piR-42773) detected by stem-loop and poly(A)-tailed RT-qPCR. (F−J) The absolute levels of 5 synthetic 2′-OH animal miRNAs (e.g., miR-16, miR-21, miR-122, miR-423, and let-7a) detected by stem-loop and poly(A)-tailed RT-qPCR. (K) The absolute levels of piRNAs (e.g., piR-31068, piR-31925, piR-43771, and piR-42773) determined by stem-loop and poly(A)-tailed RT-qPCR in seminal plasma. (L) The absolute levels of animal miRNAs (e.g., miR-16, miR-21, miR-122, miR-423, and let-7a) determined by stem-loop and poly(A)-tailed RT-qPCR in seminal plasma. Data are represented as the mean ± SEM (n = 6). **P < 0.01, *** P < 0.001.

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