Space transcriptome experiment | what factors will affect the quality of space transcriptome sequencing during the preparation of clinical tissue samples?

Spatial transcriptomics is considered as an important component of spatiotemporal molecular images , Molecular information can be linked to clinical images . High quality human sample preparation and processing will ensure the generation of accurate and reliable information from clinical spatial transcriptome .

In recent days, , From Zhongshan Hospital Affiliated to Fudan University 、 The scientific research teams of Shenzhen Huada Institute of life sciences and other institutions are in 《Clinical and Translational Medicine》 Publish the latest research results , The aim was to identify the possible effects of spatial transcriptome sequencing on lung cancer 、 The quality of space transcriptome in adjacent tissues or normal tissues 、 Section pathological images and RNA Potential factors for completeness .

Research ideas

The study included 57 For lung cancer 、 Of adjacent or normal tissue 125 Samples , The discovery study collected 33 For the organization 77 Samples , The validation study collected 24 For the organization 48 Samples . use Stereo-seq technology Spatial transcriptome sequencing . The research team classified the possible influencing factors from the clinical aspect , Include patient selection 、 Pathological definition 、 Type of surgery 、 Sample collection 、 Temporary storage conditions and solutions 、 Freezing method 、 Transportation and storage conditions and time . The histological score was mainly studied （histological scores） and RNA Full value (RIN) And unique molecular identifier (UMI) The relationship between . 

Research process

The main results are as follows

This study investigated the potential factors that may affect the quality of spatial transcriptomics in lung cancer , Before spatial transcriptome sequencing , Through the spatial transcriptome HE Dyeing quality and RIN The value is certain RNA Integrity to evaluate the quality of pathological images .RIN Recommended as a definition RNA One of the best methods for integrity quality , It is also the first step to evaluate gene expression . However , The results did not show histological scores >90 or RIN And UMI A significant relationship between , This may mean a histological score >90 Does not affect the quality of the spatial transcriptome samples , The number of samples is small ,RIN Also not shown with UMI of .

therefore , The research team tried to figure out the location of the organization 、 Transportation and storage 、 Delivery time of samples 、 Sample handling method 、-80℃ Storage days and other potential factors . The results show that , The effects of sample separation time and drying conditions on the spatial transcriptome samples UMI And quality are essential , It is suggested that the temporary storage time and conditions of samples in the operating room should be within 30 Within minutes , be in “ dry （dry）” state . It is recommended that human lung samples be directly cryopreserved at the optimal cutting temperature (OCT) In the medium .

Sample collection and processing HE and RIN The factors influencing the score

Quality control of space transcriptome sequencing

Discuss

The selection of spatial transcriptome samples is highly dependent on the understanding of clinicians and patients 、 Nature and severity of the disease 、 Pathological size and location . Histopathological diagnosis and local lesion confirmation are also the quality assurance to ensure that spatial transcriptomics produces accurate information .

The storage time of samples in operating room and cryopreservation time are indirect factors , and The transport mode from operating room to laboratory is a direct factor affecting the quality of space transcriptome , This study shows that Sample storage time in operating room >30 Minutes and cryopreservation time >7 days , The quality of tissue morphology is low . Use RNA The impact of in situ hybridization with probes needs to be further verified in large-scale populations . When the quality of histomorphology is low , It is difficult to determine the location and distribution of positive staining .

The changes of temporary storage conditions and solutions directly affect the quality of tissue morphology and spatial transcriptome . Dry ice and OCT To prepare lung tissue , At the same time, the research data show that , Before sequencing, the samples were in -80℃ The storage time of should be kept at 7 Within days . When the sample is temporarily stored in dry ice and transferred to -80°C when ,RNA The degradation rate can be effectively reduced .

Histological quality and RIN Binding is a predictor of spatial transcriptome . Before conducting spatial transcriptome studies , It is recommended that HE Score and RIN Values , To present the morphological integrity and rRNA The integrity of , And it is defined in the display method of spatial transcriptomics .

Besides , There are other challenges to be faced and overcome in the comprehensive preparation of clinical samples of spatial transcriptome . For example, how to OCT Filling or penetrating the alveolar space of excised human tissue ; sample RNA The degree of damage to integrity is highly correlated with the number of times the sample is repeatedly frozen and thawed ; Another challenge is to select and define the ideal slice location 、 angle 、 integrity 、 shape 、 Size and thickness , To obtain the best features of spatial molecular images and positions ; The annotation of clustering is also a challenge .

# CNGBdb It can provide the storage service of spatial omics data

The spatial transcriptome data of the above studies have been uploaded to the national gene bank sequence archiving system (https://db.cngb.org/cnsa/)

reference

Liu X, Jiang Y, Song D, et al. Clinical challenges of tissue preparation for spatial transcriptome[J]. Clinical and Translational Medicine, 2022, 12(1): e669.

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