Protocol for qPCR analysis that corrects for cDNA amplification efficiency

Damgaard, Mads V.; Treebak, Jonas T.

doi:10.1016/j.xpro.2022.101515

Cited by 17 publications

(13 citation statements)

References 3 publications

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“…This cannot be completely avoided during primer design process and highly depends on the qPCR machine, the choice of reagents for PCR, the presence of various inhibitors in the sample and even the sample volume used for the dilutions 26 . Therefore, it is important to correct the real-time PCR data using primer amplification efficiency to enhance the analytical accuracy 12 . We added this correction to our integrity value calculations ensuring a robust method for RNA integrity assessment.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, it is important to correct the real-time PCR data using primer amplification efficiency to enhance the analytical accuracy 12 . We added this correction to our integrity value calculations ensuring a robust method for RNA integrity assessment.…”

Section: Discussionmentioning

confidence: 99%

“…Next, a linear regression curve was generated and the slope of the trend line was calculated. Amplification efficiency was estimated using the following equation: Then the amplification factor was calculated and corrections for the expression were made as described by Damgaard and Treebak 12 .…”

Section: Methodsmentioning

confidence: 99%

“…Also, no such assay for mice or human RNA samples was developed. Furthermore, recent studies demonstrated that even minor differences of primer efficiencies result in significant under- or overestimation of mRNA levels, which should be accounted for in a qPCR based assay 12 .…”

Section: Introductionmentioning

confidence: 99%

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Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations

Bujanauskiene,

Merkevicius,

Kuliesiute

et al. 2024

Preprint

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Traditionally, RNA integrity evaluation is based on ribosomal RNAs (rRNAs). Nevertheless, gene expression studies are usually focused on protein coding messenger RNAs (mRNAs). As rRNA and mRNA have significant structural and functional differences, the assumption that rRNA integrity properly represents mRNA integrity may not be accurate. Moreover, contrary to whole tissue RNA samples, subcellular preparations such as synaptosomes contain almost no rRNA, thus prohibiting the use of traditional rRNA-based methods to assess sample RNA integrity. Here we present a RT-qPCR based assay, which estimates mRNA integrity by comparing the abundance of 3 prime and 5 prime end mRNA fragments in a long constitutively expressed mouse or human PGK1 mRNA. The assay was tested and validated using plasmids with cloned 3 prime and 5 prime ends of the PGK1 cDNA reflecting different ratios of 3 prime and 5 prime cDNA amplicons in partially degraded RNA samples. The accuracy of integrity score calculation was ensured by integrating a mathematical correction of qPCR results to account for the variable amplification efficiency of different primer pairs. The 5 prime:3 prime assay was used to quantify RNA degradation in heat-degraded mouse and human brain tissue RNA as well as in clinical human brain RNA samples. Importantly, the expression of housekeeping genes correlated better with 5 prime:3 prime integrity value than with the RIN. Finally, we were even able to use 5 prime:3 prime assay to assess mRNA integrity in mouse synaptosomal preparations that lack rRNAs. We concluded that the 5 prime:3 prime assay can be used as a reliable and sensitive method to evaluate mRNA integrity in mouse and human brain tissue and subcellular preparations.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations

Bujanauskiene,

Merkevicius,

Kuliesiute

et al. 2024

Preprint

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“…[ 27 ] Increasing NAD + by administering NR was also shown to prevent NIHL and ARHL in rodents, [ 18,28 ] but the instability of NR in the circulation complicates its delivery and limits its treatment efficacy, which might be the reason for suboptimal clinical efficacy in human with oral NR supplementation. [ 29 ] Another potential NAD + booster with potential efficacy in treating NIHL is NAM, which is both an NAD + precursor and a byproduct of NAD + consumption. Chronic dose‐appropriate supplementation of NAM, which can be converted to NAD + through the salvage pathway, has been shown to improve healthspan [ 30 ] and ameliorate neurodegeneration in the central and peripheral nervous systems.…”

Section: Introductionmentioning

confidence: 99%

Personalized Porous Gelatin Methacryloyl Sustained‐Release Nicotinamide Protects Against Noise‐Induced Hearing Loss

Feng,

Dong,

Song

et al. 2024

Advanced Science

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There are no Food and Drug Administration‐approved drugs for treating noise‐induced hearing loss (NIHL), reflecting the absence of clear specific therapeutic targets and effective delivery strategies. Noise trauma is demonstrated results in nicotinamide adenine dinucleotide (NAD+) downregulation and mitochondrial dysfunction in cochlear hair cells (HCs) and spiral ganglion neurons (SGNs) in mice, and NAD+ boosted by nicotinamide (NAM) supplementation maintains cochlear mitochondrial homeostasis and prevents neuroexcitatory toxic injury in vitro and ex vivo, also significantly ameliorated NIHL in vivo. To tackle the limited drug delivery efficiency due to sophisticated anatomical barriers and unique clearance pathway in ear, personalized NAM‐encapsulated porous gelatin methacryloyl (PGMA@NAM) are developed based on anatomy topography of murine temporal bone by micro‐computed tomography and reconstruction of round window (RW) niche, realizing hydrogel in situ implantation completely, NAM sustained‐release and long‐term auditory preservation in mice. This study strongly supports personalized PGMA@NAM as NIHL protection drug with effective inner ear delivery, providing new inspiration for drug‐based treatment of NIHL.

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