Oocytes were extracted in M2 media (Sigma; M7167) plus 2.5 mM Milrinone (Sigma; M4659) to maintain GV arrest. The surrounding somatic cells were removed by pipetting. The oocytes were treated briefly with Tyrode’s solution (Sigma; T1788) to remove the zona pellucida. Between 50 to 150 denuded GV oocytes were added to 350 μL RLT buffer from the QIAGEN RNAeasy kit (QIAGEN; 74134). The samples were processed according to the QIAGEN protocol and the RNA sample was eluted in 30 μL H2O. Half of the sample was used to produce cDNA using the Promega cDNA kit (Promega; M3682), together with oligo(dT)15 (Promega; C1101). To normalize the samples, the volume of cDNA was diluted according to the initial cell number. The qPCR was carried out in duplicates using SYBR Green (Thermo Scientific; 4309155). Rsp16 was used as a house-keeping gene to normalize the data [44 (link)]. The Smc1βprom-smc1α transgene negative sample was set to the value of 1 in each case. Primers were:
Cdna kit
Manufactured by Promega
Sourced in United States
The cDNA kit is a laboratory product used for the reverse transcription of RNA into complementary DNA (cDNA). This process is a fundamental step in gene expression analysis, allowing researchers to study and quantify the expression of specific genes.
Automatically generated - may contain errors
Lab products found in correlation
Oligo dt 15, by Promega (2 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Biorobot ez1, by Qiagen (2 mentions)
Milrinone, by Merck Group (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
M2 media, by Merck Group (1 mentions)
Tyrode s solution, by Merck Group (1 mentions)
Rneasy mini kit reagent, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Glutathione s transferase gst, by Biodiagnostic (1 mentions)
Glutathione peroxidase gpx, by Biodiagnostic (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Microalbumin assay kit, by Abcam (1 mentions)
Hyperoside, by Merck Group (1 mentions)
Cd206, by Thermo Fisher Scientific (1 mentions)
Foxp3, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
8 protocols using cdna kit
1
Transcriptome Analysis of Oocyte GV Arrest
2
Quantification of UUAGGG Repeat Transcripts
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RNA was reverse transcribed to cDNA using the Promega cDNA kit (M3682; Promega), together with oligo(dT)15 (C1101; Promega). To amplify UUAGGG repeat transcripts, we used CCCTAA-specific oligos for generation of cDNA for qPCR analysis. 1 μg of cDNA was used for qPCR analysis. qPCR was carried out in duplicates using SYBR Green (4309155; Thermo Fisher Scientific). Beta actin was used as a house-keeping gene to normalize the data.
3
Axolotl Cloacal Gland Transcriptome Analysis
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Total RNA was extracted from the axolotl cloacal gland tissue using RNeasy Mini Kit reagents (Qiagen, USA) according to manufacturer instructions. Residual DNA was removed using DNase I digestion and cDNA was constructed using the Promega cDNA kit. Briefly, total RNA (1 μg) was reverse-transcribed at 42°C into DNA using 5 U of reverse transcriptase, 1x RT buffer, 1 mM of deoxyribonucleotide triphosphate, 10 ng/μl of random primer, and 20 U RNase inhibitor in a 20 μL reaction. RNA quality and quantity were assessed with an Agilent 2100 BioAnalyzer. Approximately 20 μg of cDNA was used for sequence analysis via Illumina HiSeq™ 2000 platform at the Michigan State University Research Technology Support Facility (East Lansing, MI) to generate 50 bp single-end reads. The raw data from Illumina HiSeq were deposited in the NCBI Short Read Archive (SRA) database (Accession: SRR2140729).
4
Immunohistochemical and Oxidative Stress Analysis
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Absolute ethanol (EtOH) solution was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Polyclonal rabbit anti-mouse iNOS antibody (1 : 100; Cat: ab15323), monoclonal rabbit anti-human Bax antibody (1 : 300; Cat: ab32503), polyclonal rabbit anti-mouse IL-1β antibody (1 : 250; Cat: ab9722), and polyclonal rabbit anti-human CD3 antibody (1 : 100; Cat: ab5690) were purchased from Abcam Co., Cambridge, UK.
Kits for malondialdehyde (MDA), GSH, total superoxide dismutase (T.SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST) were obtained from Biodiagnostic Co. (Dokki, Giza, Egypt). Total RNA extraction and SYBR Green Master Mix kits were purchased from Qiagen Co., Germany. cDNA kit was obtained from Promega Co., Madison, WI, USA.
Kits for malondialdehyde (MDA), GSH, total superoxide dismutase (T.SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST) were obtained from Biodiagnostic Co. (Dokki, Giza, Egypt). Total RNA extraction and SYBR Green Master Mix kits were purchased from Qiagen Co., Germany. cDNA kit was obtained from Promega Co., Madison, WI, USA.
5
Detection of Paramyxovirinae in Animal Tissues
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For each animal, total nucleic acids were extracted from approximately 1 mm3 of lung, kidney and spleen using the viral mini kit v2.0 and an EZ1 BioRobot (QIAGEN). Tissues were homogenized in DMEM medium by TissueLyser (QIAGEN) for 2 min at 25 Hz using 3 mm tungsten beads. cDNA was generated through reverse transcription (Promega cDNA kit). Semi-nested PCR targeting a partial sequence of the L-gene polymerase locus (∼490 bp) of Paramyxovirinae subfamilies was carried out using previously described conditions5 (link), 49 (link). RT-PCR products were purified using the QIAquick PCR purification kit (QIAGEN), cloned into the pGEM-T Easy vector (Promega), according to the manufacturer’s instructions and sequenced using chain-termination (Sanger) sequencing (Big Dye Sequencing kit, ABI) on both strands by a commercial service (Genoscreen).
6
Micro-albumin Quantification and Cell Assay Protocol
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The micro-albumin assay kit was obtained from Abcam Biotechnology (Abcam, Cambridge, UK). Hyperoside with a purity higher than 98% was purchased from Sigma-Aldrich (St Louis, USA) and was suspended in 0.5% carboxymethyl cellulose sodium salt high viscosity (CMCNa) (MP Biomedicals, LLC, USA) solution for animal experiments or dissolved in 0.1% DMSO for cell culture experiments. Glucose was purchased from Sigma-Aldrich (St Louis, MO, USA) while Trizol reagents were manufactured by Invitrogen (California, USA). cDNA Kit was purchased from Promega (Promega Corporation, Madison, WI). 24-well transwell chamber was bought from Corning (Corning, Shenzhen, China). All flow cytometric antibodies, including F4/80, CD11b, CD86, CD206, CD4, IL4, and Foxp3 were purchased from eBioscience or Biolegend. And details of the antibodies used in this study are listed in the Table 1
7
Comprehensive Pathogen Screening Protocol
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Approximately 1 mm3 of lung, kidney, and spleen collected from the same animal were pooled in DMEM medium and homogenized in a TissueLyser II (Qiagen, Hilden, Germany) for 2 min at 25 Hz using 3 mm tungsten beads. Total nucleic acids were extracted from the mixture supernatant using the viral mini kit v2.0 and an EZ1 BioRobot (Qiagen). cDNA products were generated via reverse transcription (cDNA kit, Promega, Madison, Wisconsin, USA). PVs were detected by a semi-nested PCR targeting part of the L-gene polymerase gene, designed as to detect Respirovirus, Morbillivirus, and Henipavirus (RMH)52 (link). The 400–600 bp PCR amplified cDNAs were purified using the Qiagen PCR purification kit and cloned into the pGEM-T vector system (Promega). Cloned PCR products were sequenced by the Sanger method (Big Dye sequencing kit, ABI, Genoscreen, Lille, France) using M13 standard sequencing primers.
8
Quantifying Transcripts from 3T3-L1 Cells
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We isolated messenger RNA (mRNA) from 3T3‐L1 cells, cultured it with L‐14 extract using the PureLink™ RNA minikit (Invitrogen) and reverse‐transcribed it into complementary DNA (cDNA) using a cDNA kit (Promega). Next, the cDNA was amplified using a TB green mix (TAKARA) using the StepOnePlus™ Real‐Time polymerase chain reaction (PCR) System (Applied Biosystems) and analysed. The primers used for quantitative reverse transcription PCR (qRT‐PCR) were designed as the listed sequences (Table S1 ).
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