Utilizing an RNA extraction kit (OMEGA Bio-Tek), whole RNA was extracted from ovarian tissue taken from the tumor and healthy surrounding tissues. Utilizing an Agilent 2,200 TapeStation and a NanoDrop, RNA integrity and purity were assessed after extraction using the following parameters: an RNA integrity number value of 7.0, a ratio of A260/A230 equal 2.0, and a ratio of A260/A280 equal 1.8.
Rna extraction kit
Manufactured by Omega Bio-Tek
Sourced in United States, China
The RNA extraction kit from Omega Bio-Tek is designed to efficiently isolate and purify RNA from a variety of sample types. The kit utilizes a simple and reliable protocol to obtain high-quality RNA suitable for downstream applications such as RT-PCR, Northern blotting, and RNA sequencing.
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Lab products found in correlation
Transstart top green qpcr supermix, by Transgene (7 mentions)
Easyscript one step gdna removal and cdna synthesis supermix, by Transgene (6 mentions)
Hiscript 1st strand cdna synthesis kit, by Vazyme (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Superscript 3 platinum one step qrt pcr kit with rox, by Thermo Fisher Scientific (2 mentions)
Agilent 2200 tapestation, by Agilent Technologies (2 mentions)
Nanodrop nd 2000, by Agilent Technologies (2 mentions)
Truseq rna sample preparation kit, by Illumina (2 mentions)
Quantstudio 5, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by GeneCopoeia (1 mentions)
Nanodrop nd 2000, by Thermo Fisher Scientific (1 mentions)
Aceq qpcr sybr green master mix, by Takara Bio (1 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Reverse transcription kit, by Promega (1 mentions)
Sybr premix ex taq reagent, by Takara Bio (1 mentions)
Abi7700 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Uv spectrophotometer, by PerkinElmer (1 mentions)
Hiscript 2 q rt supermix for qpcr kit, by Vazyme (1 mentions)
Lightcycler 480 2 real time pcr detection system, by Roche (1 mentions)
50 protocols using rna extraction kit
1
Ovarian Tissue RNA Extraction Protocol
2
TXNRD1 Knockdown Efficiency Evaluation
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In order to assess the knockdown efficiency of TXNRD1, total RNA was extracted from PASMCs using RNA Extraction Kit (#6834, OMEGA biotek) according to the manufacturer’s instructions. First-strand cDNA was reverse transcribed from total RNA using the First-Strand cDNA Synthesis Kit (GeneCopeia). mRNA level of TXNRD1 was quantified by Real-time PCR using SYBR Green qPCR Mix Kit (GeneCopeia) on QuantStudio 5 (Thermo) with following primer: 5′-GGTGAAAGGCCGCGCTA-3′ (forward), 5′ATAGGACGCGCCAACCACTA-3′ (reverse). Data were analyzed using the 2-ΔΔCT method with GAPDH as an internal control.
3
Comparative Transcriptional Analysis of Trichoderma reesei Strains
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Fresh mycelia of T. reesei TRB1 and T.reesei RUT-C30 cultivated under different conditions were prepared and the total RNA was extracted with the RNA extraction Kit (Omega Bio-Tek, Inc, USA) following the manufacturer’s protocol. The first-strand cDNA was synthesized from RNA using HiScript 1st Strand cDNA Synthesis Kit (Vazyme, China). Reverse transcription (RT) was performed using the AceQ qPCR SYBR Green Master Mix (Takara, Dalian, China). The reversed RNA concentration was determined at 260 nm using a NanoDrop ND-2000 (Thermo Fisher Scientific, Wilmington, DE). qRT-PCR was performed on the ABI StepOne instrument Plus (ABI, Germany) with software Version 2.3 (ABI, Germany). The primers for qRT-PCR are shown in Additional file 1 : Table S1. At least three biological triplicates were performed, and qRT-PCR of each gene was performed in three triplicates. The expression of pgk1 was chosen as the reference gene for data normalization.
4
Seedling RNA Extraction and cDNA Synthesis
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Total RNA was extracted from the seedling shoots of WYG and osprpl18-1 at the third-leaf stage using the RNA extraction kit (Omega Bio-tek, R6830). First strand cDNA was synthesized with random hexamers or RT primer mix using the PrimeScript II 1st strand cDNA Synthesis Kit (Takara, 6210A). For RT-PCR analyses, primers were designed situated on exons flanking with the concerned introns. For qRT-PCR, primers were designed positioned across intron/exon junctions of the indicated transcripts. Some primers used in this study were consistent with that in previous research (Lv et al., 2020 (link)).
5
Quantitative RT-PCR Analysis of EBV Transcripts
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Total RNA was extracted and purified and used to synthesize cDNA. An RNA extraction kit (Omega Bio-Tek Inc., Doraville, GA, uSA) was used to extract total RNA from induced tumor tissues and matched lymphocytes from 'normal' donors, with B 95-8 cells as a positive control (B 95-8 cells were derived from marmoset leukocytes transformed by EBV). The quality and quantity of the RNA samples were determined using a uV spectrophotometer (Perkin-Elmer, Fremont, CA, USA), followed by storage at -80˚C. In accordance with the instructions of the reverse transcription kit (Promega Corporation, Madison, WI, USA), 2 µg RNA from each sample was used for cDNA synthesis. mRNA expression was detected by qRT-PCR using SYBR Premix Ex Taq reagent (Takara, Dalian, China). qRT-PCR was carried out in a 96-well plate using an ABI 7700 Real-Time PCR system (Applied Biosystems, Foster City, CA, uSA). The primers used are listed in Table I.
RNA from B 95-8 cells was used as a template to obtain cDNA for qRT-PCR. A standard curve was established based on the initial cDNA duplication number and CT value for real-time fluorescent quantitative detection of B 95-8 cells. The template content in the sample was determined by relative quantitation using the ΔΔCT method, standardized by the duplication number of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. Each sample was tested in triplicate.
RNA from B 95-8 cells was used as a template to obtain cDNA for qRT-PCR. A standard curve was established based on the initial cDNA duplication number and CT value for real-time fluorescent quantitative detection of B 95-8 cells. The template content in the sample was determined by relative quantitation using the ΔΔCT method, standardized by the duplication number of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. Each sample was tested in triplicate.
6
Transcriptomic Analysis of Tomato Fruit
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Total RNA was extracted from the tomato fruit powder using the RNA extraction kit (Omega Bio Tek, Norcross, GA, USA) following the manufacturer’s instructions. 0.5 μg of total RNA was reverse transcribed into cDNA using a HiScript II Q RT SuperMix for qPCR Kit (Vazyme Biotech, Nanjing, China). The qRT-PCR experiments were conducted on the Light Cycler® 480 II Real-Time PCR Detection System (Roche, Swiss). The tomato housekeeping gene ACTIN (Solyc03g078400) was used as internal control to calculate the relative expression of target genes. PCR was performed with 3 min at 95 °C, followed by 45 cycles of 30 sec at 95 °C, 30 sec at 58 °C, and 1 min at 72 °C. Sequences of primers are listed in Table S1 .
7
RNA-Seq Analysis Pipeline for Cell Transcriptomes
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Total RNA was extracted using an RNA extraction kit (Omega Bio‐Tek, Norcross, GA, USA), and then, quantitative RNA sequencing was performed by BGI (Beijing, China) using the Ion Torrent platform. The raw reads were first filtered using soapnuke (v1.5.2) software (BGI) with the parameters: ‐l 15 ‐q 0.5 ‐n 0.1 to remove the low‐quality reads (> 20% of the base qualities were < 10), reads with adaptors and reads with unknown bases (N bases > 5%). Clean reads were then assembled into Unigenes, followed with Unigene functional annotation, SSR detection and calculated the Unigene expression levels and SNPs of each sample. The clean reads were mapped using HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts) to the reference genome hg38 downloaded from the University of California, Santa Cruz (http://genome.uscs.edu/ ). The gene expression levels were quantified using the software package Sailfish. The reads per kilobase of exon model per million reads method was used to calculate the gene expression level. The differential expressed genes (DEGs) between the S‐ and N‐type cells were determined by the analysis method based on a Poisson distribution. The genes with an expression log2 value larger than 2 and a P‐value of < 0.05 were filtered and considered as significant DEGs.
8
RNA Extraction and Sequencing Protocol
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Total RNA was obtained from cells by an RNA extraction kit (Omega Bio-Tek, Norcross, USA), according to the manufacturer’s instructions. RNA quality and quantity were determined on a nanophotometer (IMPLEN, GmbH, Germany). Then, equal amounts from each of the four replicates were pooled together for RNA sequencing.
9
Oroxin B Induces Autophagy Inhibition
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Oroxin B (OB, HY-N1435, purity: 99.71%, CAS number: 114482-86-9), Dimethyl sulfoxide (DMSO, HY-Y0320, purity: ≥99.0%, CAS number: 67-68-5), and autophagy inhibitor 3-methyladenine (3-MA, HY-19312, purity: 99.83%, CAS number: 5142-23-4) were acquired from MedChemExpress (Monmouth Junction, NJ, USA). Recombinant mouse IL-1β was purchased from R&D systems (Minneapolis, USA). Boster (Wuhan, Hubei, China) provided the RIPA lysis buffer, protease inhibitors, phosphatase inhibitors, BCA protein assay kit, protein loading buffer, rabbit/mouse secondary antibodies, and DAPI reagent. The ECL chemiluminescent substrate was obtained from Bio-Rad (Hercules, CA, USA). The biotechnology of Biosharp Life Sciences (Hefei, Anhui, China) provided the Trypsin (BL527A) and collagenase II (BS164). DMEM/F12 medium culture was got from Hyclone (Logan, UT, USA). Foetal bovine serum (FBS) was purchased from BioInd (Biological Industries, Israel). Omega Bio-tek (USA) provided the RNA extraction kit (R6834-01). Yeasen (Shanghai, China) provided the kits for complementary DNA (cDNA) synthesis (11141ES60) and quantitative real-time polymerase chain reaction (RT-qPCR) (11201es08).
10
RNA Extraction and qRT-PCR Analysis
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Following the manufacturer’s instructions, total RNA was extracted using an RNA extraction kit (Omega Bio-Tek, Shanghai, China) and reverse transcribed into cDNA using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China). The qRT-PCR was carried out on a Bio-Rad CFX platform (Bio-Rad, Hercules, CA, USA) with TransStart® Top Green qPCR SuperMix (Transgen, Beijing, China) using the following program: 95 °C for 30 s, then 40 cycles at 95 °C for 10 s and 60 °C for 30 s, followed by melt curves at 65 °C for 10 s and 0.5 °C increments to 95 °C. We selected the reference β-actin gene as an internal standard for normalization [22 (link)]. Supplementary Table S2 contains a list of the primers used in the qRT-PCR.
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