Total RNA was isolated and purified from genomic DNA using the NucleoSpin® RNA XS kit (Macherey Nagel). To harvest cells from the lung-on-chip, an aliquot of 100 μL lysis buffer was applied to every well, pipetted up and down three times, and transferred to a separate tube. RNA concentration and purity was analyzed on a NanoDrop Lite (Thermo Scientific). Purified total RNA was used for cDNA preparation with the Super Script III Reverse Transcriptase kit (Life Technologies) employing random hexamer primers. cDNAs were amplified by quantitative real-time PCR using SYBR® Select Master Mix (Thermo Scientific) on a 7500 Fast Start Real-Time PCR system (Applied Biosystems). Primers for ENACα (Cat.no: QT0002883) and Caveolin1 (Cat.no: QT00012607) were purchased from Qiagen. The primer sequences for 18S and hSP-C are provided in Table S1 .
7500 fast start real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7500 Fast Start Real-Time PCR system is a powerful instrument designed for real-time PCR analysis. It provides rapid, accurate, and reliable results for a wide range of applications, including gene expression analysis, pathogen detection, and genetic research. The system features a high-performance optical detection system and advanced thermal cycling technology to deliver optimal performance and efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Faststart universal sybr green master mix, by Roche (2 mentions)
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Nanodrop lite, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna xs kit, by Macherey-Nagel (1 mentions)
Dna free dna removal kit, by Thermo Fisher Scientific (1 mentions)
Deep sequencing, by Illumina (1 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (1 mentions)
3 protocols using 7500 fast start real time pcr system
1
Lung-on-Chip RNA Isolation and qPCR Analysis
2
Quantitative RT-PCR for Transcriptome Analysis
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Total RNA was isolated using the TRIzol reagent (Invitrogen). After DNAse digestion of total RNA (DNA‐free DNA Removal kit, Ambion, Thermo Fisher Scientific, Waltham, MA, USA), complementary DNA was synthesized with random hexamer primers using Super Script III Reverse Transcriptase (SuperScript® III First‐Strand Synthesis System, Invitrogen). mRNA was analysed by quantitative reverse transcription PCR with a 7500 Fast Start Real‐Time PCR system (Applied Biosystems Foster City, CA, USA) using the FastStart Universal SYBR Green Master Mix (Roche, Basel, Switzerland). Transcript levels were normalized to 18S ribosomal RNA levels. Primer sequences are available upon request.
3
Quantitative Transcriptome Analysis of Muscle Tissues
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Total RNA was isolated from muscle tissues using the TRIzol reagent (Invitrogen). For quantitative reverse transcription (qRT)-PCR, total RNA was subjected to DNAse digestion followed by complementary DNA preparation with random hexamer primers using Super Script III Reverse Transcriptase (Invitrogen). Messenger RNA (mRNA) was analyzed by quantitative real-time PCR using the FastStart Universal SYBR Green Master Mix (Roche) with a 7500 FastStart Real-Time PCR system (Applied Biosystems). Transcript levels were normalized to 18S ribosomal RNA. Primer sequences are provided in ESM 1 : Table S6. miRNA levels were measured using qRT-PCR with the TaqMan miRNA assays (Applied Biosystems). The ubiquitous miR-let-7 was used as an endogenous control based on its stable expression in all samples and its conservation between mice and humans. For mRNA and small RNA profiling, RNA was subjected to Illumina deep sequencing using a service from LC Sciences (Houston, TX, USA). The sequence results were obtained as FPKM (fragment per kilobase of exons per million reads) for each transcript.
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