The methods of RNA extraction from cells and collagen discs, and cDNA conversion, were based on a previous study [55 (link)]. Briefly, total RNA was extracted from the cells using NucleoSpin RNA (Macherey Nagel GmbH & Co., KG, Duren, Germany) and from the collagen discs using ISOGEN reagent (Nippon Gene, Tokyo, Japan), following the manufacturer’s instructions. Subsequently, 1 μg of total extracted RNA was converted into cDNA using ReverTra Ace reverse-transcription reagents (TOYOBO, Osaka, Japan), following the manufacturer’s instructions. qPCR was performed using gene-specific PrimeTime qPCR probes (Integrated DNA Technologies, Coralville, CA, USA) and Thunderbird SYBR qPCR mix (TOYOBO), according to the manufacturer’s instructions. The expression levels of the target genes were normalized to those of vimentin. The primer sequences are listed in Table S5 .
Isogen reagent
Manufactured by Nippon Gene
Sourced in Japan
ISOGEN reagent is a mono-phasic solution containing phenol and guanidinium isothiocyanate. It is designed for the isolation of high-quality total RNA from a variety of biological samples, including cells, tissues, and microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (27 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (26 mentions)
Rneasy mini kit, by Qiagen (16 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (15 mentions)
Thermal cycler dice real time system, by Takara Bio (14 mentions)
Thunderbird sybr qpcr mix, by Toyobo (13 mentions)
Revertra ace qpcr rt kit, by Toyobo (11 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (11 mentions)
Sybr premix ex taq 2, by Takara Bio (10 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Revertra ace, by Toyobo (9 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (9 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (9 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (8 mentions)
Sybr premix ex taq, by Takara Bio (7 mentions)
Superscript 3, by Thermo Fisher Scientific (7 mentions)
Nanodrop one, by Thermo Fisher Scientific (6 mentions)
Kapa sybr fast qpcr kit, by Roche (5 mentions)
Sybr green realtime pcr master mix, by Toyobo (5 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (5 mentions)
252 protocols using isogen reagent
1
RNA Extraction and qPCR Analysis Protocol
2
RNA-seq Analysis of comPDAC-FPCL Model
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RNA was isolated from comPDAC-FPCL models on Days 0, 7, and 14 using the ISOGEN reagent (Nippon Gene), following the manufacturer’s instructions. RNA-seq was performed as previously described [55 (link)]. GO enrichment analysis elucidated the functions of the differentially expressed genes using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). A volcano plot was constructed using VolcaNoseR (https://huygens.science.uva.nl/VolcaNoseR/ (accessed on 11 April 2023)). The raw sequences in FASTQ format are available from DDBJ (DRA016977).
3
Quantitative Gene Expression Analysis of 3T3-L1 Cells
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Total RNA of cultured 3T3-L1 cells was prepared with ISOGEN reagent (Nippon gene, Toyama, Japan). Complementary DNA (cDNA) of each tissue was prepared by reverse transcriptase (RT) reactions from 1 μg total RNA using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). The sequences of the PCR primers were as follows: asct2 (forward 5′-GTC ACA GCC ACA GCA TCC A-3′, reverse 5′-CCA GCC CCA AAA GCA TCA C-3′), adipoq (forward 5′-TGG CAG AGA TGG CAC TCC TG-3′, reverse 5′-GGT CGT AGG TGA AGA GAA CG-3′), pparg1 (forward 5′-TGA AAG AAG CGG TGA AC-3′, reverse 5′-TAG TGT GGA GCA GAA AT-3′), pparg2 (forward 5′-GCT GTT ATG GGT GAA AC-3′, reverse 5′-TAG TGT GGA GCA GAA AT-3′), b-actin (forward 5′-AGC CAT GTA CGT AGC CAT CC-3′, reverse 5′-ATT ACC GAG GAC GAG CCC AGA C-3′). Quantitative mRNA expression analysis was performed by a real time PCR system (StepOneTM, Applied Biosystems Inc., Foster City, CA, USA). To estimate the level of transcripts quantitatively, b-actin transcript was used as an internal control for the each prepared sample.
4
Whole Mouse Genome Microarray Analysis
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Total RNA was isolated with Isogen reagent (NIPPON GENE CO., LTD., Tokyo, Japan) and purified with RNAeasy mini kit (QIAGEN, California, USA) according to the manufacturer's instructions. Total RNA concentration and purity were checked with NanoDrop spectrophotometer (NanoDrop Technologies Inc., Delaware, USA). The quality of total RNA was assessed by electrophoretic separation on an RNA Nano lab chip, using a 2100 Bioanalyzer (Agilent Technologies Inc., California, USA). The total RNA samples were amplified and labeled with Cy3 by using the Quick Amp labeling Kit and hybridized with an Agilent 4×44 K Whole Mouse Genome Microarray (Agilent Technologies Inc.). Then, the array was scanned with Dual-Laser microarray Scanner G2565AA (Agilent Technologies Inc.). The scanned data were analyzed using Feature Extraction Software 9.1 (Agilent Technologies Inc.), which tagged the data as signals recognized as being outliers or equal to the background. The fold change of each gene was calculated as the ratio of signal intensity between the experimental average data and the control average data.
5
Quantifying Gene Expression in Breast Cells
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Total RNA was isolated from cultured cells and normal breast tissues using Isogen reagent (Nippon Gene, Tokyo, Japan) and RNeasy Mini kits (QIAGEN, Valencia, CA, USA) according to the manufacturers' instructions. The cDNA mixture was synthesised from 1 μg total RNA by reverse-transcription using Superscript III and oligo (dT) primer (Life Technologies) according to the manufacturer's protocol. Real-time polymerase chain reaction (real-time PCR) was performed to determine the expression levels of ERO1-α, VEGF and β-actin. Expression values for each sample were normalised to β-actin, and fold levels of the indicated genes represent the mean (±s.e.m.) of replicate reactions. Primer sequences were as follows: β-actin (ACTB), Hs01060665_g1; ERO1-α (ERO1L), Hs00205880_m1; and VEGF, Hs00900055_m1 (Life Technologies). PCR cycles were performed on the StepOne Real-Time PCR System (Life Technologies) with the following cycle conditions: 2 min at 50 °C, 10 min at 95 °C, 45 cycles of 15 s at 95 °C and 1 min at 60 °C. The delta–delta Ct method was used for data analysis.
6
Cloning and Sequencing of Lamprey and Cartilaginous Fish Genes
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Previously reported partial nucleotide sequences of lamprey Pax2/5/8A and Nkx2-1/2-4 orthologues of hagfish and catshark were obtained from GenBank (Accession numbers: AB079852; AB747372; AB773852) [25 (link), 31 (link), 45 (link)]. The sequences of hagfish Pax2/5/8A, B, and Hhex, and lamprey Pax2/5/8B, and Hhex were obtained from the previously reported transcriptome data sets from the GenBank database (SRA accession numbers: SRX2541845-SRX2541849; SRX2847491-SRX2847498) [46 (link)]. The nucleotide sequences of catshark Pax2, 5, 8, and Hhex were obtained from assembled transcriptome database Squalomix (https://transcriptome.riken.jp/squalomix/ ) [47 (link)].
The total RNA was extracted from the frozen or fresh whole embryos using the ISOGEN reagent (Nippon Gene, Toyama, Japan). The fragments of each target gene amplified with KAPA TaqExtra DNA polymerase (KAPA Biosystems, Wilmington, MA, USA) using gene-specific primer sets (Additional file3 : Table S2) were cloned into the pGEM-T-Easy vector (Promega, Madison, WI, USA) and sequenced. Digoxigenin-labeled RNA probes were synthesized by using these plasmids. The plasmids containing lamprey Nkx2-1/2-4 paralogues (Nkx2-1/2-4A, B, C) generated in a previous study were used for probe synthesis [25 (link)].
The total RNA was extracted from the frozen or fresh whole embryos using the ISOGEN reagent (Nippon Gene, Toyama, Japan). The fragments of each target gene amplified with KAPA TaqExtra DNA polymerase (KAPA Biosystems, Wilmington, MA, USA) using gene-specific primer sets (Additional file
7
Total RNA Isolation and qPCR Analysis
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For total RNA preparation, 2.0×105 cells were cultured in 24-well tissue culture plates. Total RNA was isolated using the Isogen reagent (Nippon Gene, Co., Ltd., Toyama, Japan) according to the manufacturer's instructions. RNA was reverse transcribed into first-strand cDNA with an RT-PCR system kit (Takara Bio, Inc., Shiga, Japan). qPCR was performed on a Thermal Cycler Dice Real-Time System (Takara Bio, Inc.) using SYBR® Premix Ex Taq™ II (Takara Bio, Inc.) with human gene-specific primers (Table I ). Target gene expression was normalized to an internal β-actin reference and expressed in terms of fold-change relative to the control sample (30 (link)).
8
Gene Expression Analysis of Il-6, Il-1a, and p16
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Total RNA was extracted from tissues using ISOGEN reagent (Nippon Gene Co., Ltd., Japan). Gene expression was quantified using primers against Il-6, Il-1a, and p16 and the PCR master mix (Cat. No. 4352042; Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Actb was used as the control gene for normalization. The level of gene expression in the untreated young group was used as the baseline, and fold change in gene expression was defined by the 2−ΔΔCT method.
9
Cerebral Cortex RNA Extraction Protocol
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Also, to elucidate the mechanism of spatial learning and memory improvement effect of GSE administration, animals were sacrificed and the brain was carefully removed after the behavioral test. The cerebral cortex was carefully dissected, frozen in liquid nitrogen and stored at -80° C until use. 100 mg of the cerebral cortex was homogenized in 1 ml of ISOGEN Reagent (NipponGene, Tokyo, Japan) using a glass-teflon homogenizer. RNA extraction was performed according to the protocol of the ISOGEN Kit (NipponGene). Briefly, 0.2 ml of chloroform (Wako, Japan) was added to the homogenized sample and centrifuged (12000 × g, 15 min, 4° C.) to isolate and collect the aqueous phase, then 0.5 mL of isopropanol (Sigma) was added to elute the RNA. After washing with 70% ethanol, the RNA solution was dissolved in Tris-EDTA buffer solution pH 8.0 (Sigma) was quantified by using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
10
Comprehensive Gene Expression Analysis in Skin and Lymph Node Tissues
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Total RNA was isolated from homogenized skin tissue using ISOGEN Reagent (Nippon Gene, Tokyo, Japan) according to the instructions from the manufacturer. Two micrograms of RNA were used for synthesis of cDNA with a cDNA synthesis kit (Applied Biosystems, Foster, CA, USA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed for skin and lymph node tis™sues with Fast SYBR Green Master Mix in a StepOne real time qPCR system (Applied Biosystems) according to the manufacturer's instructions. The quantity of mRNA for each gene was normalized with that of the housekeeping gene ornithine decarboxylase antizyme-1 (Oaz1). The primer sequences and PCR conditions for each of the genes Ifng, Tnf, Il10, Tgfb1, Cd29, Cd90, Cd105 and Oaz1 are described in Table 1 .
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