Cel mir 39 spike in

Manufactured by Qiagen

Sourced in Germany

The Cel-miR-39 spike-in is a laboratory product used as a control in microRNA quantification experiments. It is a synthetic miRNA derived from the Caenorhabditis elegans organism, which is commonly used as an exogenous spike-in control to monitor RNA extraction efficiency and enable data normalization during quantitative reverse transcription PCR (RT-qPCR) analysis of microRNA expression.

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Lab products found in correlation

Mirneasy serum plasma kit, by Qiagen (2 mentions) Total rna purification kit, by Norgen Biotek (2 mentions) Hispec buffer, by Qiagen (1 mentions) Lightcycler 96 real time pcr system, by Roche (1 mentions) Miscript 2 rt kit, by Qiagen (1 mentions) Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions) Taqman technologies, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Cel-miR-39 spike-in control Cel-miR-39 (1.6 × 108 copies) spike-in control UniSp6/cel-miR-39 spike-in mix Spike‐in cel‐miR‐39‐3p MiRNeasy Serum/Plasma Spike-In Control (Cel-miR-39) Syn-cel-miR-39 spike-in Cel-miR-39-3p spike-in control Syn-cel-miR-39 spike in synthetic RNA Cel-miR-39

5 protocols using cel mir 39 spike in

Identifying and Validating Candidate miRNAs

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Candidate miRNAs were identified using miRDeep2 software (47 (link)). Only miRNAs supported by >5 reads were reported in this study. AGO-CLIP data were mapped to the mouse genome using STAR (same as for small RNA-seq libraries described in Data Processing) and the reads falling within the putative miRNA coordinates were counted using featureCounts. We counted a putative miRNA as “supported” if it had >5 AGO-CLIP counts.
To search for the previous mentions of identified miRNAs, we looked up their sequences in miRCarta (85 (link)) and used the Google search engine to query the literature. Candidate miRNAs were ranked by novoMiRank scores, which we computed as described in ref. 85 (link).
For independent validation, we performed RT-qPCR using custom Small RNA TaqMan probes (Life Technologies; catalog #4398987) designed on the star consensus sequence reported by miRDeep2. We used 0.5 ng of total RNA per tissue sample supplied with Cel-mir-39 spike-in (Qiagen; catalog #339390) to perform the reactions in a final volume of 20 μL.
We analyzed tissue and sex specificity of identified miRNAs based on transcripts supported by at least 50 sequencing reads across all samples. Statistical analysis and data visualization were performed as described above for annotated miRNAs.

Isakova A., Fehlmann T., Keller A, & Quake S.R. (2020). A mouse tissue atlas of small noncoding RNA. Proceedings of the National Academy of Sciences of the United States of America, 117(41), 25634-25645.

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Extraction and Quantification of Urinary miRNA

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RNA was isolated from cell-free urine using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions by the use of a QIAcube robotic workstation for automated RNA extraction. The exogenous Cel-miR-39 spike-in (Qiagen) was added in equal concentrations to all samples prior to RNA isolation to monitor the efficiency of RNA extraction. Total RNA was eluted in 15 μL of RNase-free water and stored at −80 °C until further use.
Before performing real-time PCR, 2 μL RNA from each sample were retro-transcribed using the miScript II RT Kit and HiSpec Buffer (Qiagen). cDNA was then diluted 10 folds and assayed in 10 μL PCR reactions; miR-27b-3p was assayed in duplicate by qPCR using the miScript SYBR Green PCR Kit and the following primer pair: hsa-miR-27b-3p miScript Primer Assay (cat. No. MS00031668). Amplification was performed using the LightCycler^® 96 Real-Time PCR System (Roche Diagnostics, Indianapolis, IN, USA) in 96-well plates.

Pontrelli P., Conserva F., Menghini R., Rossini M., Stasi A., Divella C., Casagrande V., Cinefra C., Barozzino M., Simone S., Pesce F., Castellano G., Stallone G., Gallone A., Giorgino F., Federici M, & Gesualdo L. (2021). Inhibition of Lysine 63 Ubiquitination Prevents the Progression of Renal Fibrosis in Diabetic DBA/2J Mice. International Journal of Molecular Sciences, 22(10), 5194.

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Urine-Derived RNA Isolation Protocol

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RNA from urine was isolated using the miRNeasy Serum/Plasma kit (Qiagen) according to the manufacturer’s instructions. In brief, 1 mL of QIAzol reagent was added to 200 µl of cell-free urine. Sample was mixed in a tube followed by the addition of 200 µl of chloroform. After vigorous mixing the sample was centrifuged at 12,000 × g for 15 minutes at 4 °C. The upper aqueous phase was carefully transferred to a new collection tube and loaded into the QIAcube robotic workstation for automated RNA extraction. The exogenous Cel-miR-39 spike-in (Qiagen, Cat No.: 219610) was added in equal amounts to all samples prior to RNA isolation to monitor the efficiency of RNA extraction. Total RNA was eluted in 15 μL of RNase-free water and stored at −80 °C until further use.

Conserva F., Barozzino M., Pesce F., Divella C., Oranger A., Papale M., Sallustio F., Simone S., Laviola L., Giorgino F., Gallone A., Pontrelli P, & Gesualdo L. (2019). Urinary miRNA-27b-3p and miRNA-1228-3p correlate with the progression of Kidney Fibrosis in Diabetic Nephropathy. Scientific Reports, 9, 11357.

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Total RNA Extraction from Adipose and Serum

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Total RNA (~20 mg of tissue) was extracted from adipose biopsies with a mirVana miRNA isolation kit (Ambion, by Life Technologies, NY, USA) according to manufacturer's protocol. One hundred μl of serum sample was used for the extraction of total RNA with Total RNA Purification kit (Norgen Biotek Corp, Thorold, Canada). The hemolysis has been controlled through visual inspection also comparing a control sample (without hemolysis evaluated by the ratio of miR-23a and miR-451) [47 (link)]. In addition, 20 fmol of spike-in cel-miR-39 (Qiagen, Hilden, Germany) was added to the serum samples at the lysis step to control for the RNA extraction efficiency. Recovery of the spike-in cel-miR-39 was constant with a mean Ct value of 16.6 ± 0.6.

Kangas R., Morsiani C., Pizza G., Lanzarini C., Aukee P., Kaprio J., Sipilä S., Franceschi C., Kovanen V., Laakkonen E.K, & Capri M. (2017). Menopause and adipose tissue: miR-19a-3p is sensitive to hormonal replacement. Oncotarget, 9(2), 2279-2294.

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Plasma RNA Extraction and miRNA Quantification

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Total RNA was extracted from plasma-EDTA samples (100 µl) with a Total RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s protocol. In addition, 20 fmol of spike-in cel-miR-39 (Qiagen, Venlo, The Netherlands) was added to the plasma samples at the lysis step as control for RNA extraction efficiency. Eight miRs were chosen, having a crucial and referenced regulatory role (see Table 1), and were measured by quantitative RT-PCR in plasma samples: miR-21-5p, -126-3p, -146a-5p, -145-5p, -133a-3p, -206, -122-5p, and -363-3p. These miRs were measured by applying TaqMan technologies (Thermo Fisher Scientific, Waltham, MA, USA); this method consists of an miR-specific retrotranscription, in which RNA is first transcribed in cDNA for each miR, then cDNA is used as a template for the quantitative PCR reaction. MiR relative expression was calculated by ΔC_t method using 2 replicates for each measurement. C_t values were normalized with miR-16-5p after validation of its stability along the time series analysis (17 (link)).

Capri M., Morsiani C., Santoro A., Moriggi M., Conte M., Martucci M., Bellavista E., Fabbri C., Giampieri E., Albracht K., Flück M., Ruoss S., Brocca L., Canepari M., Longa E., Di Giulio I., Bottinelli R., Cerretelli P., Salvioli S., Gelfi C., Franceschi C., Narici M, & Rittweger J. (2019). Recovery from 6-month spaceflight at the International Space Station: muscle-related stress into a proinflammatory setting. The FASEB Journal, 33(4), 5168-5180.

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