Nextflex small rna seq kit

Manufactured by PerkinElmer

Sourced in United States

The NEXTFLEX® Small RNA-Seq Kit is a laboratory equipment product designed for small RNA sequencing. It provides a method for preparing small RNA samples for high-throughput sequencing analysis.

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Spelling variants of this product

NEXTFLEX Small RNA-Seq Kit v3 (39 citations)

13 protocols using nextflex small rna seq kit

Profiling Podocyte Transcriptome via RNA-Seq

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RNA sequencing of the three groups of podocytes was performed, and the differentially expressed genes (DEGs) between the groups were analyzed.
Podocytes after treatment and incubation were suspended in RNAlater^® (Invitrogen, Carlsbad, CA, USA) and stored at −20 °C until further analysis to prevent RNA degradation. Total RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and DNase digestion was performed to prevent genomic DNA contamination. RNA integrity and quantity of each sample were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Using mRNA captured from total RNA by using oligo-dT beads, a library was constructed using the NEXTFLEX^® Rapid RNA-Seq Kit (PerkinElmer, Inc., Waltham, MA, USA), whereas the small RNA-Seq library was prepared using the NEXTFLEX^® Small RNA-Seq Kit (PerkinElmer Inc., Waltham, MA, USA) following the manufacturer’s instructions. Paired-end sequencing was performed using Illumina Nexseq550 (Illumina, San Diego, CA, USA) with a 100-bp read length after the quality control process, and the quantity of the libraries was assessed using the Agilent 2100 Bioanalyzer.

Lee J.M., Ko Y., Lee C.H., Jeon N., Lee K.H., Oh J., Kronbichler A., Saleem M.A., Lim B.J, & Shin J.I. (2021). The Effect of Interleukin-4 and Dexamethasone on RNA-Seq-Based Transcriptomic Profiling of Human Podocytes: A Potential Role in Minimal Change Nephrotic Syndrome. Journal of Clinical Medicine, 10(3), 496.

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FFPE Small RNA Library Preparation

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Libraries were prepared for 50 bp single-end sequencing using a NEXTflex Small RNA-Seq Kit (PerkinElmer Inc., Waltham, MA, USA). Total RNA was extracted from FFPE samples using the ReliaPrep FFPE Total RNA MiniPrep Kit (Promega, Madison, WI, USA). Small RNAs were synthesized as single-stranded copy DNAs (cDNAs) using reverse transcription priming. The quality of these cDNA libraries was evaluated using a TapeStation 4200 (Agilent, Santa Clara, CA, USA), followed by quantification using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer’s protocol. Sequencing was progressed as single-end (50 bp) using an Illumina NovaSeq6000 (Illumina, San Diego, CA, USA). The sequencing output was approximately 3 GB per sample.

Shin E., Han S.H., Park I.S., Wee J.H., Lee J.S, & Kim H. (2023). Does the Necrotic Portion of Metastatic Lymphadenopathy from Squamous Cell Carcinoma Still Have Tumoral Oncologic Information? Differential Diagnosis of Benign Necrotic Lymphadenopathy Using microRNA. Biomedicines, 11(9), 2407.

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Profiling Human Testis piRNAs

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First, sequencing reads were trimmed with Cutadapt (v.#3.0)⁴⁷ (link) according to instructions provided by CATS small RNAseq kit protocol (Diagenode, Cat. #C05010040, Doc. # v.2 I 09.17) or NEXTflex Small RNA-Seq Kit protocol (PerkinElmer, Cat. #NOVA-5132-05, Doc. # v.V19.01). Next, trimmed reads were aligned to reference genome (GRCh37) with Bowtie (v.#1.0.1)⁴⁸ (link) allowing only perfect matches, discarded miRNAs by selecting reads between 25 and 45 bases, and re-aligned to GRCh37 allowing one mismatch. Finally, known small non-coding RNAs, other than piRNAs, were removed from the dataset using DASHRv2 (v.#v2),⁴⁹ (link) and the remaining piRNA sequences were intersected with known piRNA loci detected in human adult testis.⁵⁰ (link)

Wyrwoll M.J., Gaasbeek C.M., Golubickaite I., Stakaitis R., Oud M.S., Nagirnaja L., Dion C., Sindi E.B., Leitch H.G., Jayasena C.N., Sironen A., Dicke A.K., Rotte N., Stallmeyer B., Kliesch S., Grangeiro C.H., Araujo T.F., Lasko P., D’Hauwers K., Smits R.M., Ramos L., Xavier M.J., Conrad D.F., Almstrup K., Veltman J.A., Tüttelmann F, & van der Heijden G.W. (2022). The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans. American Journal of Human Genetics, 109(10), 1850-1866.

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Small RNA Sequencing Adaptation

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In order to assay RNA features of all sizes, we adapted the NEXTFLEX Small RNA-Seq Kit protocol (PerkinElmer). Specifically, RNA was sheared naturally during the cell lysis and Strep-Tactin bead incubation stages described above (Figure S6A); and in order to include RNA fragments up to ~300 nucleotides, we omitted the upper-bound bead purification from protocol step F. Multiplexed libraries were sequenced using a version 3 paired-end 150-cycle MiSeq flow cell (Illumina).

Landeras-Bueno S., Wasserman H., Oliveira G., VanAernum Z.L., Busch F., Salie Z.L., Wysocki V.H., Andersen K, & Saphire E.O. (2021). Cellular mRNA triggers structural transformation of Ebola virus matrix protein VP40 to its essential regulatory form. Cell reports, 35(2), 108986.

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Small RNA Sequencing Adaptation

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Cardiac-Related miRNA Expression Analysis

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84 miRNA cardiac-related array analysis was performed on LightCycler 480 platform (Roche, Basel, Switzerland ) for tissue (AC1–AC9) samples and blood (AC1, AC2, AC10–AC16) samples from nine AC patients and 4 ctrl, respectively, while using MiScript miRNA PCR Array Human Cardiovascular disease (Qiagen, Hilden, Germany), according to the manufacturer’s instructions (Supplementary Material). Data analysis and normalization were performed with the GeneGlobe analysis tool (Qiagen, Hilden, Germany).
Library preparation while using TruSeq Small RNA Library Preparation kit (Illumina, San Diego, CA, USA) was used on tissue samples of three AC (AC1–AC3) probands and two ctrl whereas Next Flex Small RNASeq kit (PerkinElmer, Waltham, MA, USA was used on blood samples of 4 AC (AC1, AC2, AC10–11) probands and 4 ctrl, according to manufacturer’s instructions. Equimolar pooled samples (10pM) were sequenced on a MiSeq platform (Illumina, San Diego, CA, USA). BaseSpace Sequence Hub was used for alignment and subsequent pairwise differential expression analysis (DESeq2) and it led to the identification of DE miRNAs, precursor groups, miRNAs families, and piRNAs for each pair of sample groups (Supplementary Material).

Bueno Marinas M., Celeghin R., Cason M., Bariani R., Frigo A.C., Jager J., Syrris P., Elliott P.M., Bauce B., Thiene G., Corrado D., Basso C, & Pilichou K. (2020). A microRNA Expression Profile as Non-Invasive Biomarker in a Large Arrhythmogenic Cardiomyopathy Cohort. International Journal of Molecular Sciences, 21(4), 1536.

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miRNA Library Preparation and Sequencing

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To generate the miRNA library, samples were adjusted to include 250 ng of pure RNA in 10.5 µl. RNA was converted to a DNA library using the NEXTFLEX® Small RNA-Seq Kit (PerkinElmer Inc., MA, USA). In brief, RNA ligation was performed using the manufacturer’s recommended 3′ and 5′ ligation solution mix. Ligated RNA was reverse-transcribed to generate cDNA, which was then amplified using a PCR master mix with a unique primer for each library. The band of interest was trimmed and recovered in 300 μl of elution buffer using 10% TBE-PAGE. Bioanalyzer DNA assay (Agilent, CA, USA) was used to determine the size distribution of the library, and the concentration was determined using Qubit dsDNA HS Assay (Thermo Fisher Scientific, MA, USA). All 47 sample libraries were pooled and sequenced on an Illumina MiSeq instrument using the MiSeq Reagent Kit v3 (150 cycles) (Illumina Inc., San Diego, CA, USA).

Fakhry M., Elayadi M., Elzayat M.G., Samir O., Maher E., Taha H., El-Beltagy M., Refaat A., Zamzam M., Abdelbaki M.S., Sayed A.A., Kieran M, & Elhaddad A. (2024). Plasma miRNA expression profile in pediatric pineal pure germinomas. Frontiers in Oncology, 14, 1219796.

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Small RNA-Seq miRNA Profiling Protocol

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NEXTFLEX^® Small RNA-Seq Kit (PerkinElmer, USA; Cat. # NOVA-5132-05) was used to extract miRNA profiling. According to the manufacturer’s specifications, 700 ng of pure RNA was utilized for each library preparation input. Ligated libraries were reverse-transcribed, amplified, and given their own special barcode primer. Utilizing a 6% TBE-PAGE gel, DNA fragments ~ 150 bp (miRNA sequences plus 3′ and 5′ adaptors) were detected and subsequently processed in a 300 ul elution buffer for purification. Equimolar amounts for each final library were pooled at a final concentration of 4 nM cDNA. The Qubit dsDNA HS Assay (Thermo Fisher Scientific, USA; Cat. # Q33230) was used to measure concentration, and the Bio analyzer DNA assay (Agilent, USA; Cat. No.: 5067-1504) was used to examine the size distribution of the pooled library. At the Genomics and Epigenomics Research Program (GERP) in CCHE 57357, Egypt, a single flow cell of the Illumina MiSeq (Illumina, Inc., USA) was used to sequence the final pooled library for about 2693 miRNAs using 75-bp single-end reads.

EL-shqnqery H.E., Mohamed R.H., Samir O., Ayoub I., El-Sayed W.M, & Sayed A.A. (2023). miRNome of Child A hepatocellular carcinoma in Egyptian patients. Frontiers in Oncology, 13, 1137585.

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Small RNA Sequencing for miRNA Quantification

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EC sections were available for a subset of 174 individuals (90 cases, 84 controls) included in our current study. In order to minimize potential batch effects, cases and controls were randomly distributed across sequencing plates. Quantification of the expression of the six miRNAs of interest was based on small RNA sequencing which was performed as part of another ongoing project. To this end, total RNA, including miRNA, was purified and quantified using the same methods as described above. Library preparation and subsequent sequencing were conducted at the NGS Competence Centre at IKMB institute (Kiel, Germany). Libraries were prepared using the NextFlex Small-RNA-Seq kit (PerkinElmer, Waltham, Massachusetts), according to the manufacturer’s instructions, and sequenced on a HiSeq 4000 instrument (Illumina, San Diego, California) with 1 ×50 bp reads. Sequencing adapters were trimmed from raw reads using Flexbar v3.4.0 [17 (link), 18 (link)]. Reads were mapped and miRNAs quantified against miRBase v22.1 [19 (link), 20 (link)] using miRDeep2 [21 (link)]. Data normalization was carried out using the R package DESeq2 v1.28.1 [22 ].

Dobricic V., Schilling M., Schulz J., Zhu L.S., Zhou C.W., Fuß J., Franzenburg S., Zhu L.Q., Parkkinen L., Lill C.M, & Bertram L. (2022). Differential microRNA expression analyses across two brain regions in Alzheimer’s disease. Translational Psychiatry, 12, 352.

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Small RNA Library Preparation and Sequencing

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RNA libraries were generated using NEXTFLEX® Small RNA-Seq Kit (PerkinElmer, U.S.A.; Cat. No.: NOVA-5132-05). For each library, 400 ng purified RNA was used as an input for library preparation according to the manufacturer’s instructions. Ligated libraries were reverse transcribed and amplified with a unique barcode primer for each one. DNA fragments ∼150 bp (miRNA sequences plus 3′ and 5′ adaptors) were determined using 10% TBE-PAGE gel then retrieved in a 300-µl elution buffer for purification. The size distribution of the pooled library was checked using the Bioanalyzer DNA assay (Agilent, U.S.A.; Cat. No.: 5067-1504) and the concentration was assessed using the Qubit dsDNA HS Assay (Thermo Fisher Scientific, U.S.A.; Cat. No.: Q33230). The final pooled library was sequenced for ∼2400 miRNAs using Illumina MiSeq system (Illumina, Inc., U.S.A.).

Mousa N.O., Sayed A.A., Fahmy N., Elzayat M.G., Bakry U., Abdellatif A., Zahra W.K, & Osman A. (2021). miRNome profiling in Duchenne muscular dystrophy; identification of asymptomatic and manifesting female carriers. Bioscience Reports, 41(9), BSR20211325.

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