We extracted RNA from the middle and caudal lung lobes using an RNA isolation kit (400,800, Agilent Technologies, Santa Clara, CA) and converted it to cDNA using a reverse transcription supermix (1,708,841, Bio‐Rad Laboratories, Hercules, CA). Next, we analyzed the gene expression levels of pro‐inflammatory cytokines IL‐6 (Mm00446190_m1), TNFa (Mm00443258_m1), chemokines CCL‐2 (Mm00441242_m1), CCL5 (Mm01302427_m1), lung epithelial surfactant protein C (Sftpc (Mm00488144_m1), and secretoglobins (Scgb1a1 (Mm00442046) by RT‐PCR using TaqMan probes and primers (Thermo Fisher Scientific).
Rna isolation kit
Manufactured by Agilent Technologies
Sourced in United States, India
The RNA isolation kit is designed to extract and purify high-quality RNA from a variety of biological samples. It utilizes a spin-column-based method to efficiently capture and wash RNA molecules, ensuring their integrity for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex multiplex immunoassay, by Bio-Rad (5 mentions)
Cdna synthesis kit, by Bio-Rad (4 mentions)
Reverse transcription supermix, by Bio-Rad (2 mentions)
Taqman probes and primers, by Thermo Fisher Scientific (2 mentions)
Diff quick, by Merck Group (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Gel documentation system, by Bio-Rad (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Icycler thermal cycler, by Bio-Rad (1 mentions)
Sybr green master mix, by Agilent Technologies (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Predesigned primer probes, by Integrated DNA Technologies (1 mentions)
19 protocols using rna isolation kit
1
Cytokine and Surfactant Gene Expression
2
Comprehensive Evaluation of Influenza Pathogenesis
3
Semi-Quantitative Gene Expression Analysis
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For gene expression analysis, a semi-quantitative method was used. Total RNA was extracted using RNA isolation kit (Agilent, India) following the manufacturer’s protocol from the wheat leaves taken from different treatments at 7 dapi. First-strand cDNA synthesis was performed using 1 μg of RNA primed with oligodT using cDNA Synthesis Kit (Bio-Rad, India) according to the manufacturer’s instructions/protocols. The gene (Inducible PAL and peroxidase) expression was analyzed using gene-specific primers (Table 1 ). PCRs of 20 μl with 1.2 μl of template cDNA were performed with 3U Taq DNA Polymerase (Bangalore GeNei, India). Actin was taken as control. Thermocycling was performed using Thermal Cycler (PaqStar) with the following cycling conditions: 95°C for 4 min, 35 cycles of 94°C for 45 s, 50.7°C (PAL) or 53°C (peroxidase) or 55°C (actin) for 45 s; 72°C for 60 s, followed by final extension of 72°C for 10 min. The final product obtained with RT-PCR was separated by electrophoresis in 1.2% agarose gel in TAE buffer using gel electrophoresis apparatus (Bangalore GeNei, India) and visualization was done in gel documentation system (Bio-Rad, India).
4
Comprehensive Evaluation of Influenza Pathogenesis
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Mouse lungs were lavaged with 1 mL sterile PBS for inflammatory cell counts. Cytospin preparations of BAL fluid were stained using Diff-Quick (Sigma St. Louis, MO) and differential cell counts were conducted based on cell morphology. The superior lobe of the right lung was homogenized in sterile PBS (1ml) by mechanical grinding. The lung homogenate was use for bacterial colony counting cytokine analysis by Bio-plex Multiplex immunoassay (BioRad, Hercules, CA). Middle and inferior lobes of the right lung were snap-frozen and homogenized under liquid nitrogen for RNA extraction using an RNA isolation kit (Agilent Technologies, Santa Clara, CA). RNA analysis was performed by RT-PCR using Assay on Demand TaqMan probes and primers (Applied Biosystems, Foster City, CA). For bacterial dissemination studies, the liver, spleen, and kidneys were homogenized in PBS and plated for colony counts. Influenza burden was determined by RT-PCR for matrix protein expression as previously described56 (link).
5
Quantifying Maize Stress Response Genes
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Total RNA was extracted from maize leaf and root samples using RNA isolation kit (Agilent, USA) according to the manufacturer’s protocols at 3 and 7 DAPI. cDNA synthesis was done using cDNA synthesis kit (Bio-Rad, USA) following the manufacturer’s protocols. qRT–PCR was performed with a SYBR qPCR Mix kit (Agilent, USA) in a Bio-Rad RT-PCR (MJ MiniOpticon™). ZmACTIN (a housekeeping gene) was used to normalize all qRT–PCR data. The primer sequences used in the qPCR analysis are listed in Table 1 . The relative expression of ZmPR-1 and ZmPR-10 was calculated using the 2−ΔΔCt.
6
Quantitative RT-PCR Expression Analysis
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Total RNA from the cells was extracted using the RNA isolation kit (Agilent technologies) per the manufacturer's instructions. After removing the genomic DNA using DNase I (Ambion), 1 μg RNA was reverse‐transcribed into cDNA using a commercially available kit (Applied Biosystems). Quantitative real‐time PCR was performed with iCycler Thermal Cycler (Bio‐Rad) using 2X SYBR Green master mix (Bio‐Rad). Forty cycles were conducted as follows: 95°C for 30 seconds, 60°C for 30 seconds, preceded by 1 minute at 95°C for polymerase activation. Primer sequences for all genes we measured in this report are shown in Table 1 . Quantification was performed by the delta cycle time method, with GAPDH used for normalization.
7
Lung Immune Response Analysis
8
Transcriptional Analysis of Wheat Biotic Stress
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The qPCR analyses were performed to see the changes in the expression profile of key genes of phenylpropanoid cascade and MAPK and WRKY transcription factors along with the heteromeric G protein, Gα, and Gβ in the wheat genotypes treated with B. sorokiniana, crude toxins, and different doses of Bipolaroxin at 7 days of inoculation under glasshouse conditions. Total RNA was extracted using RNA isolation kit (Agilent, Santa Clara, CA, USA) and cDNA was synthesized using cDNA synthesis kit (BioRAD, Hongkong, China) following the manufacturers’ instructions. The quality and quantity of cDNA were analyzed using a nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). Expression analyses were performed using gene-specific primers (Supplementary Tables S2–S4 ) using sybr green master mix (Agilent, India) on the BioRAD Real Time PCR System (Model: MJ MiniOpticon, BioRAD, India) according to Singh et al. [42 (link)]. Actin was taken as the internal control. The relative transcript levels were calculated using the 2−ΔΔCT method [43 (link)].
9
EV-Associated miRNA Profiling by NGS
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To analyse RNA contents in EVs, a Qiagen RNA isolation kit was used to extract total RNA and RNA was analysed using the Agilent 2100 bioanalyser (Agilent Tech, U.S.). MiRNA profiling of EVs from normoxic and IPC HK2 cell culture medium was performed by the Professional Ribo Biotech Corporation (Guangzhou, China). Briefly, total RNA samples were fractionated on a 15% Tris-borate-EDTA (TBE) polyacrylamide gel (Invitrogen, Thermo Fisher) and small RNAs ranging between 18 and 30 nucleotides (nt) were used for library preparation. Small RNAs were reverse transcribed and amplified by polymerase chain reaction (PCR). The PCR products were sequenced using the Illumina HiSeq 2500 platform. The filtering criteria for up or downregulated genes were fold change of ≥ 2 and P < 0.05.
10
Quantitative Gene Expression Analysis
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To determine the relative gene expression level, total RNA was extracted from 100-mg plant tissue using an RNA isolation kit (Agilent Technologies), and first-strand cDNA was synthesized via reverse transcription reaction with the Revert Aid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific). Gene expression was analyzed by quantitative reverse transcription polymerase chain reaction using the LightCycler 480 Real-Time PCR System (Roche) with UBQ10 as an internal control, and gene-specific primers were shown in table S3.
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