3’ RNA sequencing for robust and low-cost gene expression profiling

Charpentier, Eric; Cornec, Marine; Dumont, Solenne; Meistermann, Dimitri; Bordron, Philippe; David, Laurent; Redon, Richard; Bonnaud, Stéphanie; Bihouée, Audrey

doi:10.21203/rs.3.pex-1336/v1

Cited by 17 publications

(21 citation statements)

References 6 publications

(7 reference statements)

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“…Samples were demultiplexed and aligned on the hg19 genome using the 3' SRP pipeline. The primary analysis of DGEseq data including, quality controls of reads, demultiplexing, read mapping, and quantification of gene expression, was carried out as described in Charpentier et al 20 Normalization of gene expression and differential expression analysis were both performed with DESeq2. 21 pvalues were adjusted with the False Discovery Rate method and genes with an adjusted p-value less than 0.05 were considered as differentially expressed (DEG).…”

Section: Discussionmentioning

confidence: 99%

Time-Limited Therapy With Belatacept in Kidney Transplant Recipients

Letellier¹,

Kervella²,

Sadek³

et al. 2022

Preprint

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In kidney transplant recipients, belatacept is usually pursued indefinitely after it has been started. In the setting of the belatacept shortage and after having evaluated the benefit-risk ratio, we established a strategy consisting of time-limited belatacept therapy / transient calcineurin inhibitor withdrawal, whose results are analyzed in that study. We considered all the kidney transplant recipients that had been switched from conventional immunosuppressive therapy to belatacept and then for whom belatacept has been withdrawn intentionally. Furthermore, in the first 8 patients, we assessed changes in peripheral blood mononuclear cells (PBMC) transcriptome using RNAseq before and 3 months after belatacept withdrawal. Over the study period, 28 out of 94 patients had belatacept intentionally withdrawn including 25 (89%) switched to low-dose CNI. One rejection due to poor compliance occurred. The eGFR after 12 months remained stable from 48 ± 19 ml.1.73m-2 to 46 ±17 ml.1.73m-2 (p = 0.68). However, patients that resumed belatacept / withdrew CNIs (n=10) had a trend towards a better eGFR comparing with the others (n =15): 54 ± 20 ml.1.73m-2 vs eGFR 43 ± 16 ml.1.73m-2 respectively (p = 0.15). The only factor associated with belatacept resumption was when the withdrawal took place during the COVID-19 outbreak. Transcriptome analysis of PBMCs, did not support rebound in alloimmune response. These findings underpin the use of belatacept as part of a time-limited therapy, in selected kidney transplant recipients, possibly as an approach to allow efficient vaccination against SARS-Cov-2.

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Section: Discussionmentioning

confidence: 99%

Time-Limited Therapy With Belatacept in Kidney Transplant Recipients

Letellier¹,

Kervella²,

Sadek³

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Retro-transcription was achieved using the cDNA Reverse Transcription High-Capacity kit (Applied Biosystem, Courtaboeuf, France). Sample preparation, the verification of sample quality and quantity, and transcriptomics were performed according to the protocol explained and detailed by Charpentier et al [ 61 ]. The identification and selection of differentially expressed genes was performed with the R package “DESeq2”, and “clusterProfiler” was used for enrichment analyses and associated graphical representation [ 62 ].…”

Section: Methodsmentioning

confidence: 99%

Beneficial Effects of O-GlcNAc Stimulation in a Young Rat Model of Sepsis: Beyond Modulation of Gene Expression

Dupas

Persello

Blangy-Letheule

et al. 2022

IJMS

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The young population, which is particularly at risk of sepsis, is, paradoxically, rarely studied. Acute stimulation of O-GlcNAcylation, a post-translational modification involved in metabolic regulation, cell survival and stress response, is beneficial in young rats with sepsis. Considering that sepsis impacts the gene expression profile and that O-GlcNAcylation is a regulator of transcription, the aims of this study are to (i) unveil beneficial mechanisms of O-GlcNAcylation and (ii) decipher the relationship between O-GlcNAcylation and transcription during sepsis. Endotoxemic challenge was induced in 28-day-old male rats using a lipopolysaccharide injection (E. coli O111:B4, 20 mg·kg−1) and compared to control rats (NaCl 0.9%). One hour after, rats were assigned to no therapy or fluidotherapy (NaCl 0.9%, 10 mL.kg−1) ± NButGT (10 mg·kg−1) to stimulate O-GlcNAc levels. Cardiac O-GlcNAcylation levels were evaluated via Western blot and gene transcription using 3′ SRP analysis. Lipopolysaccharide injection favorizes inflammatory state with the overexpression of genes involved in the NF-κB, JAK/STAT and MAPK pathways. NButGT treatment increased cardiac O-GlcNAcylation levels (p < 0.05). Yet, the mRNA expression was not impacted two hours after fluidotherapy or NButGT treatment. In conclusion, O-GlcNAc stimulation-induced beneficial effects are not dependent on the gene expression profile at the early phase of sepsis.

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“…The primary analysis of DGEseq data including, quality controls of reads, demultiplexing, read mapping, and quantification of gene expression, was carried out as described in Charpentier et al [ 20 ] Normalization of gene expression and differential expression analysis were both performed with DESeq2 [ 21 ]. p -values were adjusted with the False Discovery Rate method and genes with an adjusted p -value less than 0.05 were considered as differentially expressed (DEG).…”

Section: Methodsmentioning

confidence: 99%

Time-Limited Therapy with Belatacept in Kidney Transplant Recipients

Letellier

Kervella²,

Sadek³

et al. 2022

JCM

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Introduction: In kidney transplant recipients, belatacept is usually pursued indefinitely after it has been started. In the setting of the belatacept shortage and after having evaluated the benefit–risk ratio, we established a strategy consisting of time-limited belatacept therapy/transient calcineurin inhibitor withdrawal, whose results are analyzed in that study. Methods: We considered all the kidney transplant recipients that had been switched from conventional immunosuppressive therapy to belatacept and then for whom belatacept has been withdrawn intentionally. Furthermore, in the first 8 patients, we assessed changes in peripheral blood mononuclear cells (PBMC) transcriptome using RNAseq before and 3 months after belatacept withdrawal. Results: Over the study period, 28 out of 94 patients had belatacept intentionally withdrawn including 25 (89%) switched to low-dose CNI. One rejection due to poor compliance occurred. The eGFR after 12 months remained stable from 48 ± 19 mL.1.73 m−2 to 46 ± 17 mL.1.73 m−2 (p = 0.68). However, patients that resumed belatacept/withdrew CNIs (n = 10) had a trend towards a better eGFR comparing with the others (n = 15): 54 ± 20 mL.1.73 m−2 vs. eGFR 43 ± 16 mL.1.73 m−2, respectively (p = 0.15). The only factor associated with belatacept resumption was when the withdrawal took place during the COVID-19 outbreak. Transcriptome analysis of PBMCs, did not support rebound in alloimmune response. Conclusions: These findings underpin the use of belatacept as part of a time-limited therapy, in selected kidney transplant recipients, possibly as an approach to allow efficient vaccination against SARS-CoV-2.

show abstract

3’ RNA sequencing for robust and low-cost gene expression profiling

Cited by 17 publications

References 6 publications

Time-Limited Therapy With Belatacept in Kidney Transplant Recipients

Time-Limited Therapy With Belatacept in Kidney Transplant Recipients

Beneficial Effects of O-GlcNAc Stimulation in a Young Rat Model of Sepsis: Beyond Modulation of Gene Expression

Time-Limited Therapy with Belatacept in Kidney Transplant Recipients

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