Polyadenylation and cdna synthesis kit

Manufactured by Qiagen

Sourced in Denmark

The Polyadenylation and cDNA synthesis kit is a laboratory tool used for the addition of a poly(A) tail to the 3' end of RNA molecules and the subsequent synthesis of complementary DNA (cDNA) from the polyadenylated RNA. This kit provides the necessary reagents and protocols to perform these essential steps in the process of gene expression analysis and cDNA library construction.

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Lab products found in correlation

Lightcycler 480 real time pcr system, by Roche (1 mentions) Mircury lna microrna pcr system, by Qiagen (1 mentions) Exilent sybr green master mix, by Qiagen (1 mentions)

2 protocols using polyadenylation and cdna synthesis kit

Quantitative Assessment of microRNAs in Extracellular Vesicles

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RNA was isolated from sEVs precipitated from 2.0 mL perfusion fluid (where possible, 1.5 mL for one sample) and reverse transcribed in 50 μL reactions using the miRCURY LNA Universal RT microRNA polymerase chain reaction (PCR), Polyadenylation and cDNA synthesis kit (Exiqon, Vedbaek, Denmark). cDNA was diluted 50× and assayed in 10 μL PCR reactions according to the protocol for miRCURY LNA Universal RT microRNA PCR; each microRNA was assayed once by qPCR on the microRNA Ready-to-Use PCR, Human Panel I using ExiLENT SYBR Green master mix. Amplification was performed in a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland) in 384 well plates. Amplification curves were analyzed using the Roche LC software, both for determination of Cq (by the second derivative method) and melting curve analysis. Amplification efficiency was calculated using algorithms similar to the LinReg software. All assays were inspected for distinct melting curves and the Tm was checked to be within known specifications for the assay. Furthermore assays must be detected with 5 Cqs less than the negative control and Cq<37 to be included in the data analysis. Cq was calculated as the second derivative. Using NormFinder the best normalizer was found to be the average of assays detected in all samples. All data were normalized to the average of assays detected in all samples (average–assay Cq)

Gremmels H., de Jong O.G., Toorop R.J., Michielsen L., van Zuilen A.D., Vlassov A.V., Verhaar M.C, & van Balkom B.W. (2019). The Small RNA Repertoire of Small Extracellular Vesicles Isolated From Donor Kidney Preservation Fluid Provides a Source for Biomarker Discovery for Organ Quality and Posttransplantation Graft Function. Transplantation Direct, 5(9), e484.

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Validating miRNA Biomarkers in T2DM Diagnosis

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miRNAs short listed as differentially expressed from the discovery set were revalidated in a larger cohort comprising of 145 individuals (50% men, 50% women) with NGT (n = 49), IGT (n = 47) and T2DM (n = 49) by individual PCR assays. Total RNA from serum was extracted as described above and used for qRT-PCR by using the Universal miRCURY LNA microRNA PCR, Polyadenylation and cDNA synthesis kit II and the miRCURY LNA microRNA PCR system, Exilent SYBR green master mix, from Exiqon, on ABI 7000 Applied Biosystems thermocycler. Relative expression of microRNA expression was calculated by 2^{ˆ(-"DeltaCt")} method. We evaluated a suitable number of reference miRNAs, based on the increased expression stability [28 (link)]. However, we were unable to identify stable miRNAs that did not discriminate between male and female or that were not correlated with metabolic parameters, or that was not previously identified as relevant for the pathology in previous studies. This could be explained partly by the fact that inside the same group, women and men might differed in their level of cholesterol and triglycerides (S1 Table). Thus, in this study, data were normalised on the volume of total RNA that was used for qRT-PCR (i.e.; a fixed volume of 8 μl).

Prabu P., Rome S., Sathishkumar C., Aravind S., Mahalingam B., Shanthirani C.S., Gastebois C., Villard A., Mohan V, & Balasubramanyam M. (2015). Circulating MiRNAs of ‘Asian Indian Phenotype’ Identified in Subjects with Impaired Glucose Tolerance and Patients with Type 2 Diabetes. PLoS ONE, 10(5), e0128372.

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