A Study On The Influence Of Blood Sample Processing On RNA Sequencing Data

The successful implementation of the human genome project and its follow-up influence have made the high-throughput sequencing technology develop unprecedentedly.However,the problems it faces are increasingly prominent.High throughput sequencing data standards are not uniform,data quality is also uneven due to the impact of various technical links,and the most complicated sample preparation has the most complex impact,which has been widely recognized and increasingly concerned.Clinical detection has become the main direction of the application and development of high-throughput sequencing technology.It is more important and significant to thoroughly understand the factors that affect the quality of sequencing data.In this paper,blood,the most commonly used clinical sample,is taken as the research object,focusing on the study of blood storage time and the quality of RNA sequencing data by Peripheral blood mononuclear cells(PBMC)sample separation method.The quality and accuracy of sequencing data are explored and analyzed.Based on the analysis of differential expression genes,some genes with regular changes and related to storage time are found.This study can expand the application of high-throughput sequencing in clinical related research and promote its further development.In PBMC,by comparing the gene expression differences between different sample treatments,we found that the differences gradually expanded with the extension of storage time,but the effect on the total gene was not significant.Five genes with gradient difference over time were found in PBMC sequencing data.They are all immune related genes.These genes were not mentioned in previous studies,which suggests that we will produce bias according to the conventional biomarker screening method without paying attention to sample processing.In addition,the effect of centrifugal speed was greater than that of preservation time,and different centrifugal speed could induce differential expression.The effects of different storage time and centrifugation speed on the same sample were also analyzed,which provided a theoretical basis for how to operate and process the sample during the blood preservation period.In the whole blood,by comparing the gene expression difference of the same sample in different storage time,we found that the gene expression difference within 24 hours was not obvious.Even,the population difference is greater than the effect of storage time.However,the sequencing results of whole blood and PBMC showed that most of the differential genes were related to the function of red blood cells,which may be the background interference caused by too much weight of hemoglobin.Therefore,although it is easy to extract RNA by whole blood kit instead of traditional PBMC,the background interference related to hemoglobin gene affects the results.