Development of a 3’:5’ digital PCR assay to determine horse mRNA integrity

Cheyne, Charis Du; Chen, Yao; Craene, Jurgen De; Thas, Olivier; Spiegelaere, Ward De

doi:10.1016/j.ab.2021.114217

Cited by 3 publications

(5 citation statements)

References 19 publications

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“…In mice and humans, there are two PGK isozymes encoded by different genes: PGK1 and PGK2 but only PGK1 is expressed ubiquitously 16 . PGK1 transcripts have been previously used to measure mRNA integrity in rat toxicology studies 10 and horse samples 11 . Here we designed the qPCR primers for 5’:3’ assay based on mouse and human PGK1 cDNA to measure mRNA integrity in mouse and human RNA samples ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…This mouse and human 5’:3’ assay is based on PGK1 mRNA as it is a long constitutively expressed transcript of a highly conserved gene 16 . The PGK1 RNA was previously successfully used for designing 3’:5’ assay primers for rat and horse RNA samples 10, 11 . Several other genes have also been tested in the past.…”

Section: Discussionmentioning

confidence: 99%

“…The 3':5' assay measures the integrity of the ubiquitously expressed mRNA of choice, independently of rRNAs. By now a few versions of the 3':5' assay have been published that used different template mRNAs for different species [8][9][10][11] . However, 3':5' assays accuracy has not been sufficiently explored as the few existing studies validate the assay only by comparing 3':5' assays integrity values to RIN values.…”

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: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

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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show abstract

“…further optimized the 3′ : 5′ integrity assay to qualify mRNA with digital PCR. 406 Interestingly, Björkman et al . provided a novel approach to quantify the RNA integrity by measuring the differential amplification (ΔAmp) of an RNase resistant endogenous marker relative to a reference gene ( i.e.…”

Section: Methods and Techniques To Quantify Nuclease-mediated Degrada...mentioning

confidence: 99%

“…Cheyne et al further optimized the 3 0 : 5 0 integrity assay to qualify mRNA with digital PCR. 406 Interestingly, Bjo ¨rkman et al provided a novel approach to quantify the RNA integrity by measuring the differential amplification (DAmp) of an RNase resistant endogenous marker relative to a reference gene (i.e., 18S RNA). This assay was able to detect 99.7% degradation and to precisely follow the decay kinetics of mRNA by RNase A, heating or UV irritation.…”

Section: Capillary Electrophoresis (Ce)mentioning

confidence: 99%