Quantification of classical HLA class I mRNA by allele‐specific, real‐time polymerase chain reaction for most Han individuals

Pan, Ning; Lü, Shan; W, Wang; Miao, Fengqin; Sun, Hang; Wu, Shenshen; Ding, Nan; Qiu, Jie; Xu, Jing; Zhang, J

doi:10.1111/tan.13186

Cited by 5 publications

(7 citation statements)

References 22 publications

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“…The drawback with previous protein expression studies has been the lack of allele-specific monoclonal antibodies to recognize all HLA alleles with equal affinity. While qPCR has been adopted for determining the expression of HLA alleles, the focus has been mainly on HLA class I ( 3 , 4 , 6 , 8 ). Given the high number of known HLA alleles, qPCR requires a combination of allele-specific primers to amplify different alleles of the same gene.…”

Section: Discussionmentioning

confidence: 99%

HLA RNA Sequencing With Unique Molecular Identifiers Reveals High Allele-Specific Variability in mRNA Expression

et al. 2021

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The HLA gene complex is the most important single genetic factor in susceptibility to most diseases with autoimmune or autoinflammatory origin and in transplantation matching. Most studies have focused on the vast allelic variation in these genes; only a few studies have explored differences in the expression levels of HLA alleles. In this study, we quantified mRNA expression levels of HLA class I and II genes from peripheral blood samples of 50 healthy individuals. The gene- and allele-specific mRNA expression was assessed using unique molecular identifiers, which enabled PCR bias removal and calculation of the number of original mRNA transcripts. We identified differences in mRNA expression between different HLA genes and alleles. Our results suggest that HLA alleles are differentially expressed and these differences in expression levels are quantifiable using RNA sequencing technology. Our method provides novel insights into HLA research, and it can be applied to quantify expression differences of HLA alleles in various tissues and to evaluate the role of this type of variation in transplantation matching and susceptibility to autoimmune diseases.

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Section: Discussionmentioning

confidence: 99%

HLA RNA Sequencing With Unique Molecular Identifiers Reveals High Allele-Specific Variability in mRNA Expression

et al. 2021

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“…Taqman PCR and allele-specific real-time PCR are commonly used for ADE detection 48,49 . However, these methods suffer a major problem, i.e., it is difficult to distinguish two alleles on the same locus 22–24 . Besides, two pairs of primers are used for these methods, which cause bias due to different amplification efficiency.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, many approaches have been applied to determine ADE, such as high-throughput DNA/RNA sequencing 12–18 , DNA microarray 19–21 , Taqman PCR 22,23 , allele specific real-time PCR 24 , and digital PCR 25 . These methods lead to the detection of major genes associated with human disease, causal mutations in cis-regulatory elements, loss of function alleles, and others 26–29 .…”

Section: Introductionmentioning

confidence: 99%

Quantification of allelic differential expression using a simple Fluorescence primer PCR-RFLP-based method

Zhao

Xie

et al. 2019

Sci Rep

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Allelic differential expression (ADE) is common in diploid organisms, and is often the key reason for specific phenotype variations. Thus, ADE detection is important for identification of major genes and causal mutations. To date, sensitive and simple methods to detect ADE are still lacking. In this study, we have developed an accurate, simple, and sensitive method, named fluorescence primer PCR-RFLP quantitative method (fPCR-RFLP), for ADE analysis. This method involves two rounds of PCR amplification using a pair of primers, one of which is double-labeled with an overhang 6-FAM. The two alleles are then separated by RFLP and quantified by fluorescence density. fPCR-RFLP could precisely distinguish ADE cross a range of 1- to 32-fold differences. Using this method, we verified PLAG1 and KIT , two candidate genes related to growth rate and immune response traits of pigs, to be ADE both at different developmental stages and in different tissues. Our data demonstrates that fPCR-RFLP is an accurate and sensitive method for detecting ADE on both DNA and RNA level. Therefore, this powerful tool provides a way to analyze mutations that cause ADE.

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“…Real-time PCR has been adopted in several studies for determining the expression of HLA alleles, however, the focus has mainly been on HLA class I. [3–5,10] Given the high number of known HLA alleles, real-time PCR approach requires a combination of allele-specific primers to amplify different alleles of the same locus. Using RNAseq data of 50 individuals, we performed a high-throughput screen for HLA expression profiles of class I and class II alleles in peripheral blood samples.…”

Section: Discussionmentioning

confidence: 99%

“…[1,2] Recently a few studies reported varying expression levels of HLA alleles based on the real-time polymerase chain reaction (PCR) and the mean fluorescence intensity (MFI). [3–10] The differential expression of HLA alleles has been associated with immunologically mediated diseases, such as Crohn’s disease [11] and HIV [6,12], follicular lymphoma[7], and the outcome of HSCT through the risk of graft versus host disease (GvHD)[8,9]. In fact, incompatibilities between the donor and the recipient in HSCT have made the expression differences of HLA molecules an interesting target for finding permissive mismatches.…”

Section: Introductionmentioning

confidence: 99%

HLA RNAseq reveals high allele-specific variability in mRNA expression

Johansson

Koskela

et al. 2018

Preprint

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18The HLA gene complex is the most important, single genetic factor in susceptibility to most 19 diseases with autoimmune or autoinflammatory origin and in transplantation matching. The majority of 20 the studies have focused on the huge allelic variation in these genes; only a few studies have explored 21 differences in expression levels of HLA alleles. To study the expression levels of HLA alleles more 22 systematically we utilised two different RNA sequencing methods. Illumina RNAseq has a high 23 sequencing accuracy and depth but is limited by the short read length, whereas Oxford Nanopore's 24 technology can sequence long templates, but has a poor accuracy. We studied allelic mRNA levels of 25 HLA class I and II alleles from peripheral blood samples of 50 healthy individuals. The results 26 demonstrate large differences in mRNA expression levels between HLA alleles. The method can be 27 applied to quantitate the expression differences of HLA alleles in various tissues and to evaluate the role 28 of this type of variation in transplantation matching and susceptibility to autoimmune diseases. 29 3 30 Author Summary 31 32Even though HLA is widely studied less is known of its allele-specific expression. Due to the pivotal role 33 of HLA in infection response, autoimmunity, and transplantation biology its expression surely must play 34 a part as well. In hematopoietic stem cell transplantation the challenge often is to find a suitable HLA-35 matched donor due to the high allelic variation. Classical HLA typing methods do not take into account 36 HLA allele-specific expression. However, differential allelic expression levels could be crucial in finding 37 permissive mismatches in order to save a patient's life. Additionally, differential HLA expression levels 38 can lead into beneficial impact in viral clearance but also undesirable effects in autoimmune diseases. To 39 study HLA expression we developed a novel RNAseq-based method to systematically characterize allele-40 specific expression levels of classical HLA genes. We tested our method in a set of 50 healthy individuals 41 and found differential expression levels between HLA alleles as well as interindividual variability at the 42 gene level. Since NGS is already well adopted in HLA research the next step could be to determine HLA 43 allele-specific expression in addition to HLA allelic variation and HLA-disease association studies in 44 various cells, tissues, and diseases. 45 54 reaction (PCR) and the mean fluorescence intensity (MFI).[3-10] The differential expression of HLA 55 alleles has been associated with immunologically mediated diseases, such as Crohn's disease [11] and 56 HIV [6,12], follicular lymphoma[7], and the outcome of HSCT through the risk of graft versus host 57 disease (GvHD)[8,9]. In fact, incompatibilities between the donor and the recipient in HSCT have made 58 the expression differences of HLA molecules an interesting target for finding permissive mismatches. 59 Although currently only the qualitative HLA typing is considered in dono...

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Quantification of classical HLA class I mRNA by allele‐specific, real‐time polymerase chain reaction for most Han individuals

Cited by 5 publications

References 22 publications

HLA RNA Sequencing With Unique Molecular Identifiers Reveals High Allele-Specific Variability in mRNA Expression

HLA RNA Sequencing With Unique Molecular Identifiers Reveals High Allele-Specific Variability in mRNA Expression

Quantification of allelic differential expression using a simple Fluorescence primer PCR-RFLP-based method

HLA RNAseq reveals high allele-specific variability in mRNA expression

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