RNA was extracted from plasma samples using a commercially available kit, according to manufacturer’s (Jena Bioscience total RNA Purification kit) instructions. Reverse-transcription (first strand cDNA synthesis) of the extracted RNA was performed using random hexamer and specific primers using Script cDNA synthesis kit (Jena Bioscience), in a final volume of 20-μl. The synthesis conditions were 42o C for 10min followed by incubation at 50oC for 45min. The NS5B gene fragment located at positions 8275–8618 of the virus was then amplified using a nested PCR protocol. The PCR reaction mix constituted of 2.0 μl of the cDNA, 2.5-μl of Taq polymerase, 0.5-μl each of forward and reverse primers and 7.0-μl of nuclease-free water in a final volume of 12.5-μl reaction. Details of the primers and cycling conditions used are shown in the table 1 below. The amplified gene fragments were visualised using gel electrophoresis in 1.5% agarose gel (Forbi et al., 2012 (link)).
Total rna purification kit
Manufactured by Jena Biosciences
Sourced in Germany
The Total RNA Purification Kit is a laboratory product designed to efficiently extract and purify total RNA from a variety of sample types, including cells, tissues, and body fluids. The kit utilizes a rapid and reliable RNA extraction method to ensure high-quality, intact RNA suitable for downstream applications such as gene expression analysis, reverse transcription, and next-generation sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Gotaq pcr master mix, by Promega (7 mentions)
Script cdna synthesis kit, by Jena Biosciences (6 mentions)
Cdna archive kit, by Thermo Fisher Scientific (6 mentions)
Dnase 1, by Thermo Fisher Scientific (5 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (3 mentions)
Mmlv reverse transcription kit, by Evrogen (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
On column dnase treatment, by Thermo Fisher Scientific (1 mentions)
Kapa sybr fast qpcr kit, by Roche (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Ds 11 spectrophotometer, by DeNovix (1 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
Sensimix sybr kit, by Meridian Bioscience (1 mentions)
M mulv reverse transcriptase, by New England Biolabs (1 mentions)
Nanodrop spectrophotometer, by Implen (1 mentions)
Rnase free dnase 1, by Promega (1 mentions)
Trypsin, by PanEco (1 mentions)
42 protocols using total rna purification kit
1
Nested PCR Detection of Virus NS5B Gene
2
Gene Expression Analysis in B. distachyon
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The frozen samples were crushed with four zirconia beads (ø 2 mm) using a Shake Master Neo (BMS, Tokyo, Japan). Total RNA was extracted with a Total RNA Purification Kit (JenaBioscience, Jena, Germany) with on-column DNase treatment (Invitrogen, Carlsbad, CA, USA). RNA concentration and purity were validated with a DS-11 spectrophotometer (Denovix, Wilmington, DE, USA). cDNA was synthesized from each sample with the PrimeScript RT reagent kit with gDNA Eraser (Takara, Shiga, Japan). Gene expression analyses were performed by qRT-PCR using a KAPA SYBR Fast qPCR Kit (KAPA BIOSYSTEMS, Woburn, MA, USA) with a GVP-9600 real-time PCR instrument (Shimadzu, Kyoto, Japan). The quantification of target transcripts was performed using the GVP-9600 internal software GVP gene detection system, and the data were normalised to the BdUbi4 gene (Bradi3g04730), which has been established as a reference gene for expression studies in B. distachyon [59 (link)]. Primers used in this study are listed in Additional file 2 : Table S1.
3
Hypoxia-induced gene expression analysis
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MCF-7, Hep3B and U2OS cells were grown in 24-well plates and pre-treated with 0.1 or 1.0 µM selinexor 1 hr before normoxic (20% O2) or hypoxic (1% O2) incubation. After 4 hrs of incubation, total RNA was extracted using the total RNA Purification Kit (Jena Bioscience, Jena, Germany) according to the manufacturer’s protocol. cDNA of total RNA (100–300 ng) was synthesized with the M-MuLV reverse transcriptase (New England Biolabs, Frankfurt, Germany) and random hexamer primers (Thermo Fisher Scientific) following the instructions of the manufacturer. Quantitative RT-PCR was performed in the Eco48 qPCR System (PCRmax Limited Beacon Road, Staffordshire, United Kingdom) with 1 μL cDNA and the SensiMix SYBR Kit (Bioline, Luckenwalde, Germany) in a total volume of 12.5 μL. PCR primer sequences can be provided upon request by the authors. Expression values were normalized to relative expression of ribosomal protein L28 (RPL28) and four technical replicates were measured.
4
Gene Expression Analysis Protocol
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To measure the expression of the QS (las, and Rhl) and ETA genes, total RNA was extracted from treated and untreated bacterial cells according to the procedure designated by the manufacturer (Total RNA Purification Kit, Jena Bioscience, Germany). The remaining genomic DNA was removed by RNase-free DNase I (Promega, USA). The purity and the concentration of the extracted RNA were determined by measuring the absorbance at (260/280 nm) by Nanodrop spectrophotometer (IMPLEN).
5
Peptide Synthesis and Characterization
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l -Glutamine, fetal bovine serum, penicillin, streptomycin, amphotericin B, Hanks’ salts, trypsin, DMEM, and (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from PanEco, Moscow, Russia. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), HEPES, DMSO, d -glucose, MPP+, KCN, MPP+, l -NAME, tert-butyl hydroperoxide, and bovine serum albumin were from Sigma-Aldrich, St. Louis, MO, USA. 666-11, SB 202190, SP 600125, salirasib, FIPI, KN-93, ML-193, PSB C5, HA-1004, U-0126, KT-5720, and U-73 were from Tocris Bioscience, Bristol, UK. Total RNA Purification kit was from Jena Biosciences, Jena, Germany. MMLV reverse transcription kit and qPCR master mix qPCRmix-HS SYBR were from Evrogen, Moscow, Russia. cAMP determination kit and BrdU cell proliferation assay kit were from Abcam, Cambridge, MA, USA. DNase I was from Thermo Fisher Scientific, Waltham, MA, USA.
The peptide was synthesized by methods of classical peptide chemistry in solution using both protected and free L-amino acids, as described earlier [49 (link)]. Purity of the final compound was not less 98% (HPLC analysis,Supplementary S2 ).
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The peptide was synthesized by methods of classical peptide chemistry in solution using both protected and free L-amino acids, as described earlier [49 (link)]. Purity of the final compound was not less 98% (HPLC analysis,
6
Transcriptome Profiling of Venom Glands
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Total RNA from venom gland tissues was extracted using Trizol reagent (Life Technologies, Carlsbad, CA, USA) or a Total RNA Purification Kit (Jena Bioscience GmbH, Jena, Germany). The sample were stored at −80 °C. Subsequently, mRNA from samples was isolated using PolyAT Tract mRNA Isolation System (Promega, Madison, WI, USA) or a FastTrack MAG mRNA Isolation Kit (Invitrogen, Carlsbad, CA, USA). Both systems used MagneSphere® technology. Subsequently, mRNA was precipitated, concentrated using 3M sodium acetate and isopropanol and stored at −80 °C. Isolated mRNA was suspended in RNase-free water and sent to Macrogen Inc. Geumchun-gu, Seoul, Republic of Korea. The mRNA sequencing libraries were prepared using the TruSeq RNA sample preparation kit (Illumina Inc., San Diego, CA, USA) with selected insert sizes of 200–400 bp. Libraries were then sequenced on the Illumina HiSeq2000 platform using (2 × 100 bp) paired-end reads.
7
Total RNA Purification and cDNA Synthesis
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Total RNA purification kit, SCRIPT cDNA Synthesis kit and qPCR GreenMaster with UNG/lowROX – blue dyed as well as primers were all obtained from Jena Bioscience (Jena Germany). Materials for tissue culture which include Gentamycin (10 mg/ml) antibiotics, Dulbecco's modified Eagle's medium and fetal bovine serum (FBS) were obtained from the WHO Polio Laboratory, Department of Virology, University of Ibadan. Hep-2 cell line used in the study was obtained from Infectious Disease Unit of Luxembourg Institute of Health. Fluorochrome-conjugated antibodies used for immunophenotyping analysis were produced by Biolegend, eBiosciences, BD Biosciences and Beckon Dickinson Co., and donated freely by Prof. Ross M. Kedl of the Department of Immunology and Microbiology, University of Colorado Denver, Aurora Colorado. 70 μM wire mesh MACS Smart Strainer was a product of Miltenyi Biotec GmbH (Bergisch Gladbach, Germany) while a hand-operated 7 ml Dounce Homogenizer used in the study was manufactured by Thomas Scientific, Swedesboro NJ, USA. Unless stated otherwise, other standard chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO).
8
Quantitative RT-PCR Analysis of Gene Expression
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A total RNA purification kit (Jena Bioscience, Munich, Germany) was used to extract the total RNA from treated and control samples. The cDNA archive kit (Applied Biosystems, Foster City, CA, USA) was used to convert RNA to cDNA. qPCR reactions contained 1 μL of cDNA, 25 μL of GoTaq PCR master mix (Promega Co., Madison, WI, USA), 0.25 μL of CXR reference dye, 1 μL of forward and reverse primers (Table 3 ), and DNase-free water to 50 μL. qPCR was performed using a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and all analyses were done in triplicate. Results were reported as relative expression levels after being normalized to GAPDH using the 2−ΔΔCt approach [48 (link)]. Primer gene sequences were taken from the NCBI database. Primers were designed and their specificities were checked using Primer3Plus and Blast, respectively [49 (link)].
9
Glucose Homeostasis Regulation Assay
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Tetrachloroauric acid and streptozotocin (Sigma-Aldrich, USA), glucose GOD-PAP (Fortress diagnostics, UK), total RNA purification kit (Jena Bioscience, Germany), PrimeScript™ RT master mix (Takara, Japan), BlasTaq™ 2X qPCR MasterMix (Applied Biological Materials, Canada), protein quantification kit BCA assay, (Abbkine, China), phosphoenolpyruvate carboxykinase activity assay kit (MyBioSource, USA).
10
Total RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis
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Total RNA purification kit (Jena Bioscience, Germany)
was used to obtain Total RNA from the cultured cells. In
the next step, DNase I (Fermentas, USA) treatment was
performed to remove DNA contamination. After that,
RevertAid First Strand cDNA Synthesis kit (Thermo
Scientific, USA) was used to synthesize cDNA. Next,
quantitative reverse transcription polymerase chain
reaction (qRT-PCR) was carried out in duplicate using
RealQ Plus Master Mix Green (Ampliqon, Denmark).
Condition of the reaction was performed as follow: 95˚C
for 10 minutes followed by 40 cycles of denaturation at
95˚C for 30 seconds, annealing at 60˚C for 30 seconds,
and extension at 72˚C for 30 seconds. The sequences
of primer sets are presented in Table 1. Specificity of
qRT-PCR products was confirmed by melting curve
analysis as well as the electrophoresis of 1.5% agarose
gel (Genfanavaran, Iran) stained with Safe stain (Yekta
Tajhiz Azma, Iran).
was used to obtain Total RNA from the cultured cells. In
the next step, DNase I (Fermentas, USA) treatment was
performed to remove DNA contamination. After that,
RevertAid First Strand cDNA Synthesis kit (Thermo
Scientific, USA) was used to synthesize cDNA. Next,
quantitative reverse transcription polymerase chain
reaction (qRT-PCR) was carried out in duplicate using
RealQ Plus Master Mix Green (Ampliqon, Denmark).
Condition of the reaction was performed as follow: 95˚C
for 10 minutes followed by 40 cycles of denaturation at
95˚C for 30 seconds, annealing at 60˚C for 30 seconds,
and extension at 72˚C for 30 seconds. The sequences
of primer sets are presented in Table 1. Specificity of
qRT-PCR products was confirmed by melting curve
analysis as well as the electrophoresis of 1.5% agarose
gel (Genfanavaran, Iran) stained with Safe stain (Yekta
Tajhiz Azma, Iran).
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