Biopsies were snap frozen in liquid nitrogen and grinded using mortar and pestle. RNA extraction was performed using AllPrep DNA/RNA Mini Kit (Qiagen) following the manufacturer’s protocol. Quality of the eluted RNA was checked in Nanodrop Spectrophotometer. One μg of total RNA was used for cDNA synthesis using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). The product was subsequently diluted and around 10ng was finally used for each reaction. Transcripts were quantified using a 7900HT Fast Real-Time PCR system (Applied Biosystems) using Taqman probe-primers sets purchased from Applied Biosystems (LCE3A Hs00820288_s1, LCE3B Hs04193180_s1, LCE3C Hs00708773_s1, LCE3D Hs00754375_s1, LCE3E Hs01631234_sH and GAPDH Hs02758991_g1). All values were normalized to the expression of the housekeeping gene GAPDH.
Taqman probe primers sets
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan probe/primers sets are a set of reagents used in quantitative real-time PCR (qPCR) experiments. They consist of a target-specific oligonucleotide probe and forward and reverse primers. The probe is labeled with a reporter dye and a quencher dye, which allows for the detection and quantification of the target DNA sequence during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Reverse transcription kit, by Roche (1 mentions)
Rnaesy mini kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Anti α sma antibody clone 1a4, by Merck Group (1 mentions)
Hoechst 33342, by Dojindo Laboratories (1 mentions)
4 protocols using taqman probe primers sets
1
Quantification of LCE gene expression
2
Kidney Gene Expression Analysis
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Kidney total RNA samples were extracted from frozen kidney cortex samples using an RNaesy Mini kit (QIAGEN). cDNA samples were synthesized from RNA samples using a reverse transcription kit (Roche Applied Science). Gene expression levels were measured by real‐time PCR, using TaqMan probe/primers sets (Applied Biosystems) for each gene.
3
Evaluating α‐SMA Expression in E14C12 Cells
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E14C12 cells were cultured in medium with or without NA808 for 7 days. α‐SMA was immunostained using anti‐α‐SMA antibody clone 1A4 (Sigma‐Aldrich, St. Louis, MO) and the secondary antibody, Goat anti‐mouse IgG antibody Cy3‐conjugated. The fluorescence intensity of Cy3 was measured. Hoechst 33342 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) fluorescence intensity was measured to detect cell number change. RNA was extracted according to the protocol with a RNeasy Mini Kit (QIAGEN, Venlo, NLD). cDNA samples were synthesized from RNA samples using a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany). Gene expression levels were measured by real‐time PCR using TaqMan probe/primers sets (Applied Biosystems, Waltham, MA, USA) for each gene. The expression level of each gene was corrected for the expression level of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH).
4
Liver RNA Extraction and Gene Expression
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Liver total RNA samples were extracted from frozen liver samples using an RNaesy Mini kit according to the protocol. cDNA samples were synthesized from RNA samples using a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science). Gene expression levels were measured by real‐time PCR using TaqMan probe/primers sets (Applied Biosystems) for each gene. The expression level of each gene was corrected for the expression level of GAPDH.
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