Effective microtissue RNA extraction coupled with Smart-seq2 for reproducible and robust spatial transcriptome analysis

Miki Yamazaki,Takuya Yoda,Chikako Sakanashi,Koji Arikawa,Haruko Takeyama,Hiroko Matsunaga,Kiyofumi Takahashi,Masahito Hosokawa

doi:10.1038/s41598-020-63495-6

Abstract

Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens.

Highlights

Transcriptome analysis allows us to comprehensively characterise the transcripts present in biological tissues[1,2]
The negligibly small rate of cross-contamination in transcripts has been verified from the serially collected samples and collection buffer. By combining this technique with RNA-seq, we have demonstrated a spatial transcriptome analysis of disc-shaped areas at 100 μm-diameter resolution and 20 μm in height in mouse brain tissue. Since this system allows the collection of specific tissue areas on demand, we can analyze the link between an anatomical region and specific gene expressions
The RNA integrity number equivalent (RINe) remained at high levels (8.4 ± 0.11 and 7.9 ± 0.16, respectively). These results indicated that the RNase would decompose the RNA in tissue during incubation, but the dehydration fixation was effective to prevent RNA degradation by inactivating RNase, while stabilising the RNAs in the tissue

Summary

Introduction

Transcriptome analysis allows us to comprehensively characterise the transcripts present in biological tissues[1,2]. In order to overcome these issues, current spatial transcriptome techniques have been developed to analyse spatial and specific transcript distributions in tissues. These techniques include laser capture microdissection (LCM)-based RNA-seq[3,4], slide-based tissue-positional transcript barcoding[5,6,7], and single-cell RNA-seq with computer modeling[8,9]. We evaluated how the RNA degradation and insufficient tissue lysis effect on the sequence read proportions, detected gene numbers, and inter-sample reproducibility in RNA-seq analysis. The results of spatial transcriptomics analysis indicated that our microtissue RNA-seq procedure could be applied for analysing various tissue samples

Methods

Results

Conclusion