Total RNA was extracted from cells in culture using the RNeasy isolation kit (Qiagen). First-strand complementary DNA (cDNA) was synthesized from total RNA using the AMV reverse transcriptase (Roche) and a combination of random hexamers and polyA primers, according to the manufacturer’s instructions. Each of the target genes was amplified in real-time from cDNA using oligonucleotides specific to that gene (sequences and conditions available upon request), with 28S-RNA, Rpl27, and Rpl4 used as the normalization controls. cDNA levels were determined using SYBR green in a MyiQ rt-PCR detection system (BioRad). All samples were run in triplicate.
Myiq rt pcr detection system
Manufactured by Bio-Rad
Sourced in Belgium, Germany
The MyiQ RT-PCR detection system is a real-time PCR instrument designed for quantitative gene expression analysis. It features a high-resolution optical system and advanced temperature control to enable accurate and reliable real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Amv reverse transcriptase, by Roche (1 mentions)
Rneasy isolation kit, by Qiagen (1 mentions)
Mesa green qpcr mastermix plus, by Eurogentec (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Qbase software, by CellCarta (1 mentions)
Primers table 1, by Integrated DNA Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr mix, by Bio-Rad (1 mentions)
M mlv reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Peqgold rnapure, by Avantor (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
6 protocols using myiq rt pcr detection system
1
Quantitative Analysis of Gene Expression
2
Comprehensive Gene Expression Analysis
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CAFs were trypsinized and cell pellets were washed twice with RNase-free water. RNA was isolated with the miRNeasy kit (Qiagen, Venlo, The Netherlands), cDNA synthesis was performed with the iScript cDNA synthesis kit (Bio-Rad), and RT-qPCR analysis was performed on the MyiQ RT-PCR detection system (Bio-Rad) by using Mesa Green qPCR MasterMix Plus (Eurogentec, Seraing, Belgium), in accordance with the manufacturer's instructions. A preliminary experiment was conducted to identify three appropriate reference genes (TBP, YWHAZ, GAPDH) out of a set of ten genes by using qBASE+ software (Biogazelle, Zwijnaarde, Belgium). Primers (Biolegio, Nijmegen, The Netherlands) are displayed in Table 1 . All primers were blasted in Primer Blast (NCBI).
3
Mouse RNA Extraction and qRT-PCR
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Freshly collected tissues or cells were flash frozen in liquid nitrogen and stored at −80°C for later processing or immediately processed using an E.Z.N.A Total RNA kit II (Omega) extraction kit following the manufacturer’s instructions. The purity and concentration of the isolated RNA was assessed using a Nanodrop 2000; 260/280 ratios were approximately 2.0. The complementary DNA was synthesized with Quanta Biosciences Qscript. Quantitative real-time polymerase chain reaction was performed using SYBER green on a Bio-Rad MyiQ RT-PCR detection system (Bio-Rad). Primers (Table 1 ) from Integrated DNA Technologies were selected from literature or designed using Mouse Primer Depot (http://mouseprimerdepot.nci.nih.gov/ ) and Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ). Melt curve analysis was used to assess primer specificity, and 18S ribosomal RNA was used as the reference gene to control for total messenger RNA recovery. Gene expression levels were evaluated by the ΔΔ threshold cycle (Ct) method.
4
RNA Isolation and qRT-PCR Analysis of Hydrogels
5
Angptl4 expression in HCMECs by qRT-PCR
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Total RNA was extracted from HCMECs with Trizol reagent and converted into cDNA with the M-MLV reverse transcriptional system Invitrogen(San Diego, CA, USA) in the presence of random hexamers Invitrogen(San Diego, CA, USA). The cDNA was used for quantitative real-time polymerase chain reaction (RT-PCR) with specific gene primers as follows: Angptl4 forward, 5′-AGACACAACTCAAGGCTCAG-3′; reverse, 5′-CTCATGGTCTAGGTGCTTGTG-3′; GAPDH forward, 5′-ACATCGCTCAGACACCATG-3′; reverse, 5′-TGTAGTTGAGGTCAATGAAGGG-3′. An MYIQ RT-PCR detection system and SYBR green PCR mix (Bio-Rad) were used to carry out RT-PCR. The relative abundance of transcript was quantified by the comparative Ct method with GAPDH as an internal control.
6
Quantifying Gene Expression by qRT-PCR
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Gene expression levels were measured by real-time reverse transcription-PCR (qRT-PCR).
For this purpose, cells were homogenized in PeqGold RNA Pure (PeqLab, #30-1010) and RNA isolated according to the manufacturer's protocol. The measurement of the RNA concentration und purity was performed by the NanoDrop 1000 (Thermo Fisher Scientific, USA). For reverse transcription the MMLV reverse transcriptase kit (Invitrogen, #28025013) was used. The qRT-PCR was performed by SYBR Green SensiMixTM (Bioline, #QT615-05) and carried out on the MyIQ RT-PCR detection system (Biorad, Germany) applying a standardized protocol as published previously (Clarner et al. 2011) (link). Relative quantification was performed using the ΔCq method with hypoxanthine guanine phoshoribosyl transferase (Hprt) as a reference gene.
For this purpose, cells were homogenized in PeqGold RNA Pure (PeqLab, #30-1010) and RNA isolated according to the manufacturer's protocol. The measurement of the RNA concentration und purity was performed by the NanoDrop 1000 (Thermo Fisher Scientific, USA). For reverse transcription the MMLV reverse transcriptase kit (Invitrogen, #28025013) was used. The qRT-PCR was performed by SYBR Green SensiMixTM (Bioline, #QT615-05) and carried out on the MyIQ RT-PCR detection system (Biorad, Germany) applying a standardized protocol as published previously (Clarner et al. 2011) (link). Relative quantification was performed using the ΔCq method with hypoxanthine guanine phoshoribosyl transferase (Hprt) as a reference gene.
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