The Spatial RNA Integrity Number Assay for in Situ Evaluation of Transcriptome Quality

Overview

Journal Commun Biol

Specialty Biology

Date 2021 Jan 9

PMID 33420318

Citations 12

Authors

Linda Kvastad

Konstantin Carlberg

Ludvig Larsson

Eva Gracia Villacampa

Alexander Stuckey

Linnea Stenbeck

Annelie Mollbrink

Margherita Zamboni

Jens Peter Magnusson

Elisa Basmaci

Alia Shamikh

Gabriela Prochazka

Anna-Lena Schaupp

Ake Borg

Lars Fugger

Monica Nister

Joakim Lundeberg

Affiliations

Soon will be listed here.

Abstract

The RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows.

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References

Anders S, Pyl P, Huber W . HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics. 2014; 31(2):166-9. PMC: 4287950. DOI: 10.1093/bioinformatics/btu638. View

Mills J, Kavanagh T, Kim W, Jun Chen B, Kawahara Y, Halliday G . Unique transcriptome patterns of the white and grey matter corroborate structural and functional heterogeneity in the human frontal lobe. PLoS One. 2013; 8(10):e78480. PMC: 3808538. DOI: 10.1371/journal.pone.0078480. View

Stahl P, Salmen F, Vickovic S, Lundmark A, Fernandez Navarro J, Magnusson J . Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science. 2016; 353(6294):78-82. DOI: 10.1126/science.aaf2403. View

Jemt A, Salmen F, Lundmark A, Mollbrink A, Fernandez Navarro J, Stahl P . An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries. Sci Rep. 2016; 6:37137. PMC: 5111054. DOI: 10.1038/srep37137. View

Fernandez Navarro J, Sjostrand J, Salmen F, Lundeberg J, Stahl P . ST Pipeline: an automated pipeline for spatial mapping of unique transcripts. Bioinformatics. 2017; 33(16):2591-2593. DOI: 10.1093/bioinformatics/btx211. View

Kvastad L, Carlberg K, Larsson L, Villacampa E, Stuckey A, Stenbeck L . The spatial RNA integrity number assay for in situ evaluation of transcriptome quality. Commun Biol. 2021; 4(1):57. PMC: 7794352. DOI: 10.1038/s42003-020-01573-1. View

Kolijn K, van Leenders G . Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue. BMC Res Notes. 2016; 9:17. PMC: 4705748. DOI: 10.1186/s13104-015-1813-5. View

Ke R, Mignardi M, Pacureanu A, Svedlund J, Botling J, Wahlby C . In situ sequencing for RNA analysis in preserved tissue and cells. Nat Methods. 2013; 10(9):857-60. DOI: 10.1038/nmeth.2563. View

Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S . Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res. 2011; 39(9):e63. PMC: 3089491. DOI: 10.1093/nar/gkr065. View

10.

Lee J, Daugharthy E, Scheiman J, Kalhor R, Ferrante T, Terry R . Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat Protoc. 2015; 10(3):442-58. PMC: 4327781. DOI: 10.1038/nprot.2014.191. View

11.

Wang X, Lim H, Son A . Characterization of denaturation and renaturation of DNA for DNA hybridization. Environ Health Toxicol. 2014; 29:e2014007. PMC: 4168728. DOI: 10.5620/eht.2014.29.e2014007. View

12.

Gallego Romero I, Pai A, Tung J, Gilad Y . RNA-seq: impact of RNA degradation on transcript quantification. BMC Biol. 2014; 12:42. PMC: 4071332. DOI: 10.1186/1741-7007-12-42. View

13.

Costea P, Lundeberg J, Akan P . TagGD: fast and accurate software for DNA Tag generation and demultiplexing. PLoS One. 2013; 8(3):e57521. PMC: 3587622. DOI: 10.1371/journal.pone.0057521. View

14.

Koressaar T, Remm M . Enhancements and modifications of primer design program Primer3. Bioinformatics. 2007; 23(10):1289-91. DOI: 10.1093/bioinformatics/btm091. View

15.

Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M . The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol. 2006; 7:3. PMC: 1413964. DOI: 10.1186/1471-2199-7-3. View

16.

Chen K, Boettiger A, Moffitt J, Wang S, Zhuang X . RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science. 2015; 348(6233):aaa6090. PMC: 4662681. DOI: 10.1126/science.aaa6090. View

17.

Rodriques S, Stickels R, Goeva A, Martin C, Murray E, Vanderburg C . Slide-seq: A scalable technology for measuring genome-wide expression at high spatial resolution. Science. 2019; 363(6434):1463-1467. PMC: 6927209. DOI: 10.1126/science.aaw1219. View

18.

Wong K, Fernandez Navarro J, Bergenstrahle L, Stahl P, Lundeberg J . ST Spot Detector: a web-based application for automatic spot and tissue detection for spatial Transcriptomics image datasets. Bioinformatics. 2018; 34(11):1966-1968. DOI: 10.1093/bioinformatics/bty030. View

19.

Lubeck E, Coskun A, Zhiyentayev T, Ahmad M, Cai L . Single-cell in situ RNA profiling by sequential hybridization. Nat Methods. 2014; 11(4):360-1. PMC: 4085791. DOI: 10.1038/nmeth.2892. View

20.

Imbeaud S, Graudens E, Boulanger V, Barlet X, Zaborski P, Eveno E . Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 2005; 33(6):e56. PMC: 1072807. DOI: 10.1093/nar/gni054. View