Highly Sensitive Detection Of Multiple MRNA Splicing Variants And Preparation Of High-Throughput MRNA Sequencing Library

In cells,DNA is transcribed into precursors messenger RNA(pre-mRNA).premRNA can be alternatively spliced into mature messenger RNA(mRNA).mRNA guides the synthesis of proteins,which is the basic process of life science.In this process,alternative splicing is the main reason for protein diversity.Abnormal splicing is related to many diseases,such as Parkinson’s disease,spinal muscular atrophy and cancer.Researches have shown that a disease is the result of the combined action of multiple abnormal splicing during the alternative splicing process.Therefore,establishing a highly sensitive and specific method for simultaneously detecting multiple splicing variants is of great significance.RNA sequencing(RNA-seq)has become an indispensable tool for analyzing differential gene expression and mRNA differential splicing in the whole transcriptome.Through RNA-seq,transcriptome can be analyzed with unprecedented depth and accuracy,and thousands of new RNA transcriptional variants in mammalian tissues and organs have been discovered,which greatly promotes our understanding of mammalian gene expression regulation and network complexity.For RNA-seq,direct sequencing can lead to the loss of low abundance information,and it is generally necessary to amplify the RNA,that is library preparation.The preparation process of library directly affects the sequencing results,therefore,improving the efficiency of library preparation is of great significance for RNA-seq.In this paper,we established an analytical method based on nucleic acid amplification technology for highly sensitive,specific,and multiple detection of three mRNA splicing variants of telomerase.In order to improve the detection throughput,we established an analysis method based on the size-coding DNA probe to detect six mRNA splicing variants closely related to breast cancer.In order to improve the accuracy of Oxford Nanopore sequencing,we established a new method for constructing mRNA libraries based on Loop mediated Isothermal Amplification(LAMP)technology.(1)We have established a ligation reaction-based polymerase chain reaction(PCR)method to detect mRNA splicing variants.Using mRNA splicing variants as targets,a pair of specific DNA probes are designed at the splicing junction site,which can be ligated under the catalytic of SplintR ligase.PCR amplification of the ligation product was performed using a pair of universal primers.During the amplification process,three different fluorescent groups(FAM,VIC,NED)modified TaqMan probes were added,and can produce defined real-time fluorescence signals to achieve multiple detections.This method uses SplintR ligase that can efficiently recognize single base differences to ensure detection specificity,employs universal PCR primers to reduce amplification bias,and utilizes TaqMan probes modified with different fluorescent groups to achieve multiple detection of splicing variants.Based on those,this method has achieved high sensitivity,specificity,and multiple detection of splicing variants,with a detection limit as low as 10 aM;And this method has been successfully applied to the quantitative analysis of splicing variants in biological samples such as cervical cancer cells.(2)The spectral overlap between fluorescent groups limits the detection throughput of commercial TaqMan probes,which can simultaneously detect four targets.In order to improve the detection throughput,we established a reverse transcription polymerase chain reaction(RT-PCR)method based on size-coding DNA probe to detect mRNA splicing variants.Using mRNA splicing variants as targets,a pair of specific DNA probes PA and PB were designed at the splicing junction site for reverse transcription and extension reactions.Under the catalysis of ProtoScript II reverse transcriptase,PA as the primer,mRNA was reverse transcribed to obtain the first strand cDNA.Using the first strand cDNA as the template,PB was extended to obtain double stranded DNA under the catalysis of Phusion HotStart Flex DNA polymerase.PCR amplification of double stranded DNA was performed using a pair of universal primers,encoding probes of different lengths for different targets to obtain PCR amplification products of different lengths.The PCR products can be separated and detected by using capillary electrophoresis.This method achieves simultaneous detection of six splicing variants with a detection limit as low as 100 aM.This method uses capillary electrophoresis with single base discrimination,which is expected to achieve simultaneous detection of dozens of splicing variants.Finally,the method was successfully applied to quantitative analysis of splice variants in biological samples such as human breast cancer cells.(3)Oxford Nanopore Technology(ONT)Nanopore sequencer has been widely used due to its low instrument and sequencing cost,small instrument size,portability,and high speed for sequencing.However,its sequencing accuracy needs to be improved.LAMP products are a series of long double stranded DNA with repeative units,which are very suitable for Nanopore sequencing.The accuracy of Nanopore sequencing is greatly improved through self-correction of LAMP products.Therefore,we established a method for constructing mRNA libraries based on Multiple Annealing and Looping Based Amplification Cycles(MALBAC)combined with LAMP.Using oligo(dT)primers to reverse transcribe mRNA into cDNA,the mRNA hybridized with cDNA was hydrolyzed under the catalysis of RNase H.Using stem-loop DNA molecules with 8 random bases as MALBAC primers,a large amount of double stem-loop structured DNA was obtained by MALBAC of cDNA.The DNA as the initial template for LAMP amplification can be amplified,and the amplified products were subsequently constructed into a library for sequencing using an Oxford Nanopore sequencer.The mRNA library constructed based on MALBAC combined with LAMP was compared with the hg19 genome database,and a total of 136 genes were identified.The accuracy of sequencing was improved through self-correction of LAMP repeating sequences.