RNA was extracted using the TaKaRa kit and cDNA libraries were made for RNA. From total RNA, mRNA was filtered using the Oligotex mRNA Midi Kit (QIAGEN, GERMANY), followed by quality and quantity check using a Nano-Drop 2000 spectrophotometer (Thermo Scientific, USA). The cDNA libraries were made using the Illumina manufacturing protocol (Ahmad et al., 2021a (link)). The total mRNA was subjected to first and second strand cDNA synthesis and adapter ligation, and low cycle enrichment was achieved by the TruSeq®RNA HT Sample Prep Kit from Illumina (USA). The products were evaluated with the Agilent 2200 TapeStation and Qubit®2.0 of Life Technologies (USA). Dilutions were made to 10 pM for generating in situ clusters on HiSeq2500 pair-end flow cell. The 2 × 100 bp sequencing was performed, resulting in 60 M reads per sample. Transcriptomic de novo was done with the Trinity program using default parameters (Grabherr et al., 2011 (link)).
Oligotex mrna midi kit
Manufactured by Qiagen
Sourced in Germany, United States
The Oligotex mRNA Midi Kit is a laboratory equipment product designed for the isolation and purification of messenger RNA (mRNA) from various biological samples. The kit utilizes oligo(dT) affinity chromatography to selectively bind and extract mRNA molecules from total RNA extracts.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (18 mentions)
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Qubit 2, by Thermo Fisher Scientific (7 mentions)
Truseq rna ht sample prep kit, by Illumina (6 mentions)
Whole transcriptome analysis kit, by Illumina (6 mentions)
Smart cdna library construction kit, by Takara Bio (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Agilent 2100, by Agilent Technologies (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Qubit 2, by Agilent Technologies (2 mentions)
Myiq2 two color real time pcr detection system, by Bio-Rad (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Sybr green supermix, by Bio-Rad (2 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Bp clonase 2 enzyme, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1, by Takara Bio (1 mentions)
Generacer kit, by Thermo Fisher Scientific (1 mentions)
40 protocols using oligotex mrna midi kit
1
Illumina-Based RNA Sequencing Pipeline
2
Construction of cDNA Library from VK2/E6E7 Cells
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The ORF of TvAP33 was cloned and inserted into the bait vector pDHB1 to form pDHB1-TvAP33. The cDNA library of VK2/E6E7 cells was constructed according to the instructions of the SMART cDNA Library Construction Kit (TaKaRa, Clontech Laboratories, CA, USA). In brief, RNase-free DNase I (TaKaRa, Clontech Laboratories) was used to remove the genomic DNA contamination from the prepared RNA samples. The mRNA was then isolated using the Oligotex mRNA Midi Kit (Qiagen, Hilden, Germany). The purified mRNA was used to synthesize double-stranded cDNA, and both ends of the cDNAs were equipped with connectors for recombination into the prey vector. The cDNAs were purified and cloned into the pray plasmid pPR3-N with the BP Clonase® II enzyme (Thermo Fisher Scientific). These recombinant plasmids were electroporated into E. coli DH10B, according to the following electrotransfer procedure: voltage 1500 V, resistance 200 Ω and capacity 25 μF. These bacterial solutions were diluted and coated onto solid medium, the number of bacterial clones was counted and the capacity of the library was calculated. The plasmid DNA was randomly extracted from 24 clones and restriction digested by SfiI to confirm the size of the inserts in the clones.
3
Transcriptome Analysis of 18 Tissue Samples
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Total RNA was extracted from 18 tissues (6 samples in 3 repeats) using the TaKaRa RNA extraction kit, and cDNA libraries were prepared. The mRNA was obtained using Oligotex mRNA Midi Kit (Qiagen, Germany). The RNA quality was assessed using a Nano-Drop spectrophotometer (Thermo Fisher Scientific, United States), followed by cDNA library preparation using the Illumina protocol (Ahmad et al., 2021a (link)). The library products were evaluated with the Agilent 2200 TapeStation and Qubit®2.0 (Life Technologies, United States). The products were diluted to 10 pM for the in situ generation of clusters on HiSeq2500 pair-end flow cells, followed by pair-end sequencing (2 × 100 bp). Finally, transcriptome de novo was performed with the Trinity program using default parameters (Grabherr et al., 2011 (link)). Gene expressions were quantified using fragments per kilobase per transcript per million mapped reads (FPKM).
4
Illumina RNA-seq of Salt-Stressed Seedlings
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Using the TRIzol Reagent (Catalog No. 15596–026, Invitrogen), total RNAs were extracted from seedlings without or with salt stress treatment. Polyadenylated RNAs were isolated using the Oligotex mRNA Midi Kit (Catalog No. 70042, Qiagen). The RNA-seq libraries were constructed using the Illumina Whole Transcriptome Analysis Kit following the standard protocol (Illumina, HiSeq system) and sequenced by the Bioscience Core Facility at KAUST on the HiSeq platform to generate high-quality pair-end reads.
5
5' RACE Protocol for Transcriptome Analysis
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RNA ligase-mediated rapid amplification of cDNA ends (5′ RACE) was performed using Gene Racer Kit (Invitrogen, CA, USA) A with few modification [20 (link)]. Large-scale total RNA was extracted from different developmental stages of fruit and flower using a hot phenol method [107 ] and treated with RNAase-free DNAase (RNeasy Mini Kit, Qiagen Sciences, MD, USA). The total RNA concentration was quantified using ND-1000 v3.1.0 (NanoDrop Technologies, Wilmington, DE). Poly (A)+ mRNA was extracted by an Oligotex mRNA Midi Kit (Qiagen, CA, USA) from 500 μM of total RNA.
Ligation of RNA adapter, reverse transcription, and 5′-RACE PCR were carried out according to the manufacturer’s instruction (Gene Racer Kit, Invitrogen) with 100 nM poly(A)+ mRNA. Non-gene specific 5′-RACE products were generated by amplification with the Gene Racer 5′ Primer (5′-CGACTGGAGCACGAGGACACTGA-3′) and Gene Racer 3′ Primer (5′-GCTGTCAACGATACGCTACGTAACG-3′). Gene-specific 5′-RACE reactions were conducted with the Gene Racer 5′ Nested Primer and gene-specific primers as follows: PCR products were gel-purified, cloned into a plasmid vector using the TA TOPO PCR Cloning Kit (Invitrogen), and sequenced using the Illumina platform.
Ligation of RNA adapter, reverse transcription, and 5′-RACE PCR were carried out according to the manufacturer’s instruction (Gene Racer Kit, Invitrogen) with 100 nM poly(A)+ mRNA. Non-gene specific 5′-RACE products were generated by amplification with the Gene Racer 5′ Primer (5′-CGACTGGAGCACGAGGACACTGA-3′) and Gene Racer 3′ Primer (5′-GCTGTCAACGATACGCTACGTAACG-3′). Gene-specific 5′-RACE reactions were conducted with the Gene Racer 5′ Nested Primer and gene-specific primers as follows: PCR products were gel-purified, cloned into a plasmid vector using the TA TOPO PCR Cloning Kit (Invitrogen), and sequenced using the Illumina platform.
6
Transcriptome Analysis of Orchid Flower Tissues
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For transcriptome sequencing, the cDNA library was prepared from an equal mixture of RNAs isolated from flower buds and mature flowers of C. ensifolium ‘tianesu’. For the Digital Gene Expression (DGE) analysis, we constructed four independent cDNA libraries for the sepal, petal, labellum, and gynostemium separately. Total RNA was extracted from approximately 0.5 g of each tissue using the TRIzol reagent (Invitrogen). Individual mRNAs were purified from total RNA using the Oligotex mRNA Midi Kit (QIAGEN) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific) to generate the cDNA library according to the Illumina manufacturer’s instructions. Briefly, fragmentation buffer was added to interrupt mRNA to short fragments. Random hexamer primers were added to these short fragments to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using the SuperScript double-stranded cDNA synthesis kit (Invitrogen) and purified with a QiaQuick PCR extraction kit (QIAGEN).The double-stranded cDNA was sequenced using illumina Hiseq 2500 platform.
7
RNA-seq of Arabidopsis Seed Transcriptome
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Using the TRIzol Reagent (15596–026, Invitrogen Co., Carlsbad, CA, USA), total RNAs were extracted from 12-day-old seedlings of wild-type C24, sad1 and SAD1-OE. Polyadenylated RNAs were isolated using the Oligotex mRNA Midi Kit (70042, Qiagen Inc., Valencia, CA, USA). The RNA-seq libraries were constructed using Illumina Whole Transcriptome Analysis Kit following the standard protocol (Illumina, HiSeq system) and sequenced on the HiSeq platform to generate high-quality single-end reads of 101 nucleotides (some with 85 nucleotides due to machine failure) in length.
8
Rapid Amplification of Differentially Expressed Genes
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MGH (6×105) cells were plated in a 10 mm cell culture dish overnight prior to treatment with 6×107 cfu of lyophilized BCG at 37°C for 2 h. Total RNA was extracted using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Then, poly A+ RNA was isolated using the Oligotex® mRNA midi kit (Qiagen, Inc., Valencia, CA, USA) and converted to cDNAs with Riboclone® cDNA Synthesis system (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocols. The RDA was performed as described by Pastorian et al (15 (link)). The samples with and without BCG treatment were subjected to repeated rounds of subtractive hybridization at 1:10, 1:100 and 1:5,000 tester to driver ratios. Only products that were upregulated in the tester population were amplifiable by polymerase chain reaction (PCR). The PCR amplified products were ligated into pGEM®T Easy Vector (Promega Corporation), transformed into DH5α cells and positive clones were selected on LB agar plates with 100 µg/ml ampicillin. Plasmid DNA was isolated (Promega Corporation) and sequenced with-21M13 forward primers (5′-GTAAAACGACGGCCAGT-3′) using the BigDye version 3.0 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The sequencing results were identified using the BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi ).
9
Transcriptome Sequencing of RNA Samples
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Total RNA of 20 μg from each of the 18 RNA samples was sent to Novogene (Beijing, China) for construction of RNA-seq libraries and sequencing. The mRNA was purified from the total RNA using the Oligotex mRNA Midi Kit (Qiagen, Beijing), and assessed for quality using the Agilent Technologies 2100 Bioanalyzer (Agilent, United States). The mRNA was then broken into short fragment (approximately 300 bp). First and second strand complementary DNA (cDNA) was synthesized using a cDNA Synthesis System kit (TOYOBO, Japan) using random hexamer-primer, following the manufacturer’s protocol. Then double-strand cDNA were purified and adapters were ligated to the short fragments. The constructed RNA libraries were sequenced on the Illumina HiSeqTM 2000 platform in paired-end (PE) mode.
10
Transcriptome Analysis of Spleen Immunity
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To survey the gene expression profile in the spleens of large yellow croakers induced by poly(I:C), a paired-end library was constructed according to the manufacturer's protocol. Polyadenylated RNA was isolated using the Oligotex mRNA Midi Kit (Qiagen Inc., Valencia, CA, USA). Two hundred nanograms of mRNA were used for the library preparation. The RNA-seq library was constructed using Illumina Whole Transcriptome Analysis Kit following the standard protocol (Illumina GA II Sequencing System). An 80 bp paired-end run was performed on the Illumina GAII platform (Illumina, Inc., San Diego, CA, USA) to assemble the whole transcriptome de novo. The generated sequence data have been submitted to the NCBI SRA database, and the accession number is SRP035897.
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