Total RNA from PBMCs was isolated using Fenozol (A&A Biotechnology). A NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific) was used to assess the RNA concentration and quality. For reverse transcription, 1 µg of total RNA, oligo(dT) primer (Promega), and M-MLV reverse transcriptase (Promega) were used. Real-time PCR was carried out using Sybr Green Master Mix (A&A Biotechnology) and QuantStudio Real-Time PCR System (Applied Biosystems). Gene expression was normalized to elongation factor-2 (EF2), and then, the relative transcript level was quantified by the method. The primer sequences (Genomed/Sigma) and annealing temperatures are listed in Table S1 (Supplementary Material, http://links.lww.com/HC9/A32 ).
Sybr green master mix
Manufactured by A&A Biotechnology
Sourced in United States
Sybr Green Master Mix is a ready-to-use solution containing Sybr Green I dye, DNA polymerase, dNTPs, and buffer components for quantitative real-time PCR (qPCR) applications. The core function of the product is to facilitate the detection and quantification of DNA amplification during the qPCR process.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (8 mentions)
M mlv reverse transcriptase, by Promega (7 mentions)
Fenozol, by A&A Biotechnology (6 mentions)
Quantstudio real time pcr system, by Thermo Fisher Scientific (3 mentions)
Oligo dt primer, by Merck Group (2 mentions)
Eco real time pcr system, by Illumina (2 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Oligo dt 15 primer, by Promega (2 mentions)
Oligo dt primer, by Promega (1 mentions)
Homogenizer, by Omni International (1 mentions)
Quantstudio 3 thermocycler, by Thermo Fisher Scientific (1 mentions)
Sybr green jumpstart taq readymix, by Merck Group (1 mentions)
Fenozol chloroform, by A&A Biotechnology (1 mentions)
Taq pcr master mix, by EURx (1 mentions)
Universal rna purification kit, by EURx (1 mentions)
8 protocols using sybr green master mix
1
Gene Expression Profiling of PBMCs
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated from LSECs after overnight culture at 37°C in an atmosphere containing 5% O2 using a modified guanidinium isothiocyanate method (Chomczynski and Sacchi, 2006 (link)). Briefly, cells were lysed in fenozol (A&A Biotechnology), and after addition of chloroform, the aqueous phase containing RNA was collected into new eppendorf tubes. Next, RNA was precipitated, centrifuged, washed in ethanol and finally dissolved in nuclease-free water. RNA concentration was measured with an ND-1000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using 0.5 μg of total RNA with random hexamers and M-MLV reverse transcriptase (Promega) according to the vendor’s instructions. Gene expression was measured by real-time PCR (Eco, Illumina) with a SybrGreen master mix (A&A Biotechnology) according to the protocol: 95°C for 5 min followed by 40 cycles of melting at 95°C – 20 s, annealing at 58–62°C – 20 s, elongation at 72°C – 30 s. The relative quantification of gene expression was calculated with the 2−ΔΔCt method. Primer sequences, annealing temperatures and length of PCR products are listed in Table 1 .
3
Quantitative RT-PCR for Zebrafish Embryos
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For RNA isolation, ~ 10 zebrafish embryos were collected in fenozol (A&A Biotechnology), frozen and stored at -80 °C. Then, the embryos were homogenized using a homogenizer (OMNI International), and total RNA was extracted using the phenol–chloroform method. cDNA was synthesized using M-MLV reverse transcriptase (Promega), and quantitative real-time PCR was performed with SYBR Green Master Mix (A&A Biotechnology) and a QuantStudio3 thermocycler (Thermo Fisher Scientific). Rps11, eef1, acbt2 and rpl13a were used as a reference genes49 (link),50 (link) The primer sequences are listed in Supplementary Table S1. To validate specificity of the qRT-PCR reaction, melt curve analysis was performed at the end of each assay. Agarose gel electrophoresis was performed to ensure presence of a single product of predicted length at the end of the qRT-PCR reaction (Supplementary Fig. S3d).
4
Quantitative RNA Analysis from Liver Tissue
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Total RNA was isolated from liver tissue using a modified guanidinium isothiocyanate method [55 (link)]. Briefly, fragments of frozen liver tissue were lysed in fenozol (A&A Biotechnology; Gdansk, Poland; 203‐50), and after addition of chloroform (POCH; Gliwice, Poland; 234431116), the aqueous phase containing RNA was collected into new Eppendorf tubes. Next, RNA was precipitated, centrifuged, washed in ethanol (POCH; Gliwice, Poland; 396420113), and finally dissolved in nuclease‐free water (Sigma; W4502). The concentration of RNA was assessed by a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific; Waltham, MA, USA). Reverse transcription was performed using 1 µg of total RNA, oligo(dT) primer (Sigma) and M‐MLV reverse transcriptase (Promega; Madison, WI, USA; M170B). Real‐time PCR was carried out using Sybr Green Master Mix (A&A Biotechnology; 2008‐100A) and QuantStudio Real‐Time PCR System (Applied Biosystems; Waltham, MA, USA). Gene expression was normalized to elongation factor‐2 (EF2), after which the relative level of transcripts was quantified by a 2−ΔΔCT method. Sequences of primers (Genomed/Sigma) and annealing temperatures are listed in Table S2 . The primers were designed to cross an exon‐exon boundary to allow the specific amplification of cDNA targets.
5
RNA Extraction and Real-Time PCR
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For RNA isolation, tumor samples were frozen in liquid nitrogen immediately after collection and stored at -80 °C until analysis. RNA isolation and qRT-PCR were performed as described previously [24 (link)]. RNA from cells was isolated using the Fenozol-chloroform method (A&A Biotechnology, Gdynia, Poland). Real-time PCR was performed using SYBR Green Master Mix (A&A Biotechnology, Gdynia, Poland) for analysis of mouse transcripts and with SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich) for analysis of human transcripts. The sequences of the primers (Sigma-Aldrich) are listed in Table S1 (Additional file 1 ).
6
Quantitative RNA Extraction and Analysis
7
Validation of Transcript Levels by qRT-PCR
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We validated the selected transcripts by qRT-PCR. Total RNA was isolated from PURO, MCPIP1 and D141N cells from three independent experiments with the guanidium isothiocyanate (GTC) method as described.. Subsequently, 1 μg of total RNA was reverse-transcribed with oligo (dT15) primers (Promega) and M-MLV reverse transcriptase (Promega). The cDNA was diluted 5-times and real-time PCR was carried out using Eco Real-Time PCR System (Illumina) with the SYBR Green master mix (A&A Biotechnology) and primers that are listed in Supplementary Table 5 . The relative levels of the transcripts were determined relative to RPS13 (ribosomal protein S13) by ΔΔCT method.
8
Quantification of Gene Expression in Lung Tissues
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Total RNA from cultured cells and tissues was isolated using the Universal RNA Purification Kit (EURx). RNA from total mice lungs were isolated using Fenozol (A&A Biotechnology). The quantity of ribosomal RNA and DNA contamination was examined using electrophoresis in 1% denaturing formaldehyde gel. Concentration of total RNA was assessed using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using 1 mg of total RNA, oligo(dT) 15 primer (Promega) and M-MLV reverse transcriptase (Promega). Realtime PCR was carried out using SybrGreen Master Mix (A&A Biotechnology) and Eco Real-Time PCR System (Illumina). For the examination of mice lung metastasis, specific probes for human GAPDH and mouse GAPDH (Life Technologies) were used with Taq PCR Master Mix (EURx). Gene expression was normalized to elongation factor-2 (EF2). mRNA level in each sample was analyzed in duplicates. The relative level of transcripts was quantified by the DDC t method. Sequences of primers (Genomed) and annealing temperatures are listed in Table S1 in Supplementary Material.
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