Total RNA was extracted from treated cells using Trizol reagents (Invitogen, Carlsbad, CA, USA), as described in the manufacturer’s protocol, and reverse transcripted to construct the complementary cDNA by a cDNA synthesis kit (Applied Biosystems, Carlsbad, CA, USA). cDNA was then diluted for real time polymerase chain reaction (PCR) analysis by using 7900HT (Applied Biosystems) and EvaGreen real time PCR supermix (Bio-rad). The following primers were used: runt-related transcriptional factor 2 (Runx2, 57 °C): forward-gTCAgCAAAgCTTCTTTTgg and reverse-TTgTTgCTgTTgCTgTTgTT; Collagen (Col-1α1, 58 °C): forward-AATggTgCTCCTggTATTgC and reverse-ggCACCAgTgTCTCCTTTgT; and β-actin (56 °C): forward-AAgAgCTATgAgCTgCCTgA and reverse-TggCATAgAggTCTTTACgg. The concentrations of unknown samples were calculated by fitting the Ct value to the standard curve by the SDS software package (Applied Biosystems).
Sds software package
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SDS software package is a tool designed to manage and access safety data sheets (SDS) for various chemical substances and products. Its core function is to provide users with quick and easy access to relevant safety information, including hazard classifications, handling instructions, and emergency procedures. The software serves as a centralized repository for maintaining and organizing SDS data.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Quantifast sybr green pcr kit, by Qiagen (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
7700 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Abi 7500 system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq pcr kit, by Takara Bio (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Oligo dt 12 18 primer, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Abi 7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
First strand cdna synthesis kit, by Cytiva (1 mentions)
5 protocols using sds software package
1
Quantitative Gene Expression Analysis
2
Quantitative Gene Expression Analysis
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Expression analyses were performed using QuantiFast Sybr Green PCR kit (Qiagen, Milano, Italy), following the one-step protocol: cDNA was first synthesized from 60 ng total RNA, and then amplified by means of the Sybr Green QuantiTect Primer (Qiagen, Milano, Italy): hENT1 (QT010000083), CHOP (QT00082278), MRP1 (QT00061159) and DKC (QT00000392). Reactions were set up in 96-well plates and loaded onto 7700 Real-Time PCR System (Applied Biosystems, Foster City, CA). Optical data obtained were analyzed using the SDS software package (version 1.9.1; Applied Biosystems, Foster City, CA). Expression levels of target gene were obtained using the comparative method of relative quantification, after normalization for the housekeeping control gene Glyceraldehyde-3-phosphate dehydrogenase GAPDH (Sigma Aldrich, Milano, Italy), as previously performed [20 (link)].
3
Quantitative Real-Time PCR Analysis
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Total RNA was isolated from cells with TRIzol reagent according to the manufacturer’s instructions. β-actin was used as an internal control. Real-time PCR was performed on an ABI 7500 System (Applied Biosystems, United States) by using the SYBR Premix Ex Taq™ PCR kit (TaKaRa, Dalian, China). The reverse transcription reaction conditions were as follows: 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min. Real-time PCR data were analyzed using the 2−ΔΔCT method with the SDS Software package (Applied Biosystems). PCR primers were shown in Table 1 .
4
Quantification of Kidney Cortex mRNA
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Total RNA isolation of kidney cortex and cell samples was performed by using TRIzol® Reagent (Life Technologies) following the manufacturer’s instructions. RT-PCR was performed by using M-MLV Reverse Transcriptase (Life Technologies) with oligo(dT)12-18 primers (Thermo Scientific, Waltham, MA, United States). Real-time PCR was carried out in 20 μL reaction mixture containing 10 μL Power SYBR® Green PCR Master mix (Applied Biosystems), 0.5 μL cDNA template, and 250 nM of each primer using the ABI 7900HT Fast Real-Time PCR system (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. The primer sequences were the same as previously described (Cao et al., 2018 (link)). The relative quantity of mRNA was calculated using the SDS software package (Applied Biosystems, Carlsbad, CA, United States) by fitting the Ct value to the standard curve and each gene expression was normalized by its own GAPDH expression level.
5
Transcriptional Analysis of EPOR in db/db Mice
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Gastrocnemius muscle tissues were isolated from db/db mice (4 and 8 week treatment). Total RNA from muscle tissue and cells was isolated using Trizol reagent (Invitrogen) according to manufacturer's instruction. Total RNA was quantified by absorbance at 260 nm. EPOR Primer F 5' CCGTGCGTTTCTGGTGTTC 3', Primer R 5' ATGCGGTGATAGCGAGGAG 3' was used to detect EPOR transcripts. mRNA was extracted using the RNAeasy mini kit (QIAGEN Science, Germantown, MD) and cDNA was synthesized from 1.5μg RNA with the first-strand cDNA synthesis kit (Amersham, Buckmgahamshire, UK). Each sample was run and analyzed in triplicate. Real-time PCR data were analyzed using the 2-ΔΔCT method with the SDS Software package (Applied Biosystems).
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