RT-PCR technique and its applications.
State-of the-art

Malewski, Tadeusz; Malewska, Alicia; Rutkowski, Raphael

doi:10.22358/jafs/67719/2003

Cited by 2 publications

(1 citation statement)

References 45 publications

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“…Unfortunately, in most of the studies, cDNA is prepared using kits containing a mixture of oligo(dT) and/or random hexamer primers [14,23,24], where oligo(dT) is not functional for lncRNA molecules without a polyA tail. It is well known that specific primers decrease background priming, but random and oligo(dT) primers maximize the number of mRNA molecules that can be analyzed from a small sample of RNA [25]. We observed that artificially adding polyA tails and then LincRNA-RoR NRON NTT using annealing anchor (dT) adapters (analogs of oligo(dT) primers) can help to quantify lncRNAs more precisely.…”

Section: Discussionmentioning

confidence: 99%

Quantification of long non-coding RNAs using qRT-PCR: comparison of different cDNA synthesis methods and RNA stability

et al. 2021

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Introduction: Long non-coding RNAs (lncRNAs), a class of regulatory RNA molecules, are over 200 nucleotides long and could be used as a new potential biomarker, but their detection methods such as qRT-PCR are still not validated, and the influence of RNA degradation on lncRNA quantification is not clear. In this study, commercially available cDNA synthesis kits were tested and the influence of RNA degradation was compared. Material and methods: Total RNA from FaDu cells was isolated and high quality RNA and highly degraded RNA samples were used. Reverse transcription was performed using three different commercially available kits and quantifications were performed using lncRNA Primer Plate and SYBR Green I Master by LightCycler 96. qRT-PCR was performed using three different cDNA samples and results are presented as the mean Ct values. A p-value < 0.05 was considered to be significant. Results: Lower lncRNA Ct values (61/90; 67.78%) after qRT-PCR quantification were observed for cDNA synthesized using random hexamer primers preceded by polyA-tailing and adaptor-anchoring steps. It was observed that 9/90 (10.00%) lncRNAs were not detectable using different cDNA synthesis methods. For 75/90 (83%) lncRNAs, RNA degradation weakly influenced lncRNA Ct values and no differences were observed between high quality RNA and degraded samples. Seventy percent of examined lncRNAs showed significantly different Ct values depending on RNA degradation. Conclusions: cDNA synthesis kits with random hexamer primers preceded by polyA-tailing and adaptor-anchoring steps allows enhancement of lncRNA quantification specificity and sensitivity. In most cases degradation of RNA samples does not affect lncRNA quantification because these molecules have good stability.

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Section: Discussionmentioning

confidence: 99%

Quantification of long non-coding RNAs using qRT-PCR: comparison of different cDNA synthesis methods and RNA stability

et al. 2021

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Development of an Assay to Quantify Rumen Ciliate Protozoal Biomass in Cows Using Real-Time PCR

Sylvester

Karnati

et al. 2004

The Journal of Nutrition

320

213

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Currently used microbial markers cannot distinguish protozoal nitrogen (N) from bacterial N, thus limiting research on protozoal quantification in vivo by the lack of a repeatable, accurate marker for protozoal N. We report the development of a real-time PCR assay targeting the gene encoding 18S rDNA to quantify the amount of protozoal biomass in ruminal fluid and duodenal digesta. Protozoal cells were harvested from rumen fluid and concentrated for evaluation of recovery of rDNA in samples from the rumen and the duodenum. The DNA from concentrated cells was extracted with virtually 100% efficiency both before and after column purification. After serial spiking of protozoal cells into duodenal fluid over the entire range of quantification, the recovery was highly linear and constant at 81%. After serially spiking increasing quantities of protozoal rDNA into a constant volume of duodenal samples, nonlinear regression verified constant recovery of background rDNA in duodenal samples regardless of the ratio of target:nontarget rDNA. Recommendations for the procedure, including replication per sample, are described herein.

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RT-PCR technique and its applications. State-of the-art

Cited by 2 publications

References 45 publications

Quantification of long non-coding RNAs using qRT-PCR: comparison of different cDNA synthesis methods and RNA stability

Quantification of long non-coding RNAs using qRT-PCR: comparison of different cDNA synthesis methods and RNA stability

Development of an Assay to Quantify Rumen Ciliate Protozoal Biomass in Cows Using Real-Time PCR

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