Selection of appropriate reference genes for normalization of qRT-PCR based gene expression analysis in SARS-CoV-2, and Covid-associated Mucormycosis infection

  1. Sunil Kumar
  2. Ayaan Ahmad
  3. Namrata Kushwaha
  4. Sheetal Negi
  5. Kamini Gautam
  6. Anup Singh
  7. Pawan Tiwari
  8. Rakesh Garg
  9. Richa Agarwal
  10. Anant Mohan
  11. Anjan Trikha
  12. Alok Thakar
  13. Vikram Saini

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Abstract

Selection of reference genes in quantitative PCR is critical to determine accurate and reliable mRNA expression. Despite this knowledge, not a single study has investigated the expression stability of housekeeping genes to determine their suitability to act as reference gene in SARS-CoV-2 or Covid19 associated mucormycosis (CAM) infections. Herein, we address these gaps by investigating the expression stability of nine most commonly used housekeeping genes including TBP, CypA, B2M, 18S, PGC-1α, GUSB, HPRT-1, β-ACTIN and GAPDH in the patients of varying severity (asymptomatic, mild, moderate and severe). We observed significant differences in the expression of candidate genes across the disease spectrum. Next, using various statistical algorithms (delta Ct, Normfinder, Bestkeeper, RefFinder and GeNorm), we observed that CypA demonstrated the most consistent expression across the patients of varying severity and emerged as the most suitable gene in Covid19, and CAM infections. Incidentally, the most commonly used reference gene GAPDH showed maximum variations and found to be the least suitable. Lastly, comparative evaluation of expression of NRF2 is performed following normalization with GAPDH and CypA to highlight the relevance of an appropriate reference gene. Our results reinforce the idea of a selection of housekeeping genes only after validation especially during infections.

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  1. ScreenIT

    SciScore for 10.1101/2022.03.15.22272441: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsConsent: We collected blood samples at the time of admission of patients to the clinic by taking informed consent.
    IRB: This study was approved by institutional Human Ethics committee All India Institute of Medical Sciences (
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was carried out to confirm primer specificity prior to synthesizing.
    https://blast.ncbi.nlm.nih.gov/Blast.cgi
    suggested: (TBLASTX, RRID:SCR_011823)
    Statistical Analysis and the application of algorithms for the selection of the most suitable reference gene: Statistical analysis was performed using modules available in GraphPad ver 5.01.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Different statistical tools namely delta Ct, Bestkeeper, Normfinder, GeNorm, and RefFinder to calculate stability of each reference gene across the disease’s severity.
    Normfinder
    suggested: (NormFinder, RRID:SCR_003387)
    GeNorm
    suggested: (geNORM, RRID:SCR_006763)
    RefFinder
    suggested: (RefFinder, RRID:SCR_000472)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2022.03.15.22272441v1 on medRxiv