Brain tissues from 11 mice after MCAO induction, 6 treated 6 hours after induction by bevacizumab, were dissected and frozen in RNAlater (Life Science Division, Sigma-Aldrich, St. Louis, MO, USA). The middle third of each hemisphere was used. The mRNA samples were analyzed in pairs per mice (injured and uninjured hemisphere) and also pooled (normal unaffected hemispheres versus stroke hemispheres, for treated and untreated groups). Total RNA was isolated using TRIzol™ reagent (Life Technologies, Invitrogen) according to the manufacturer's protocol and then reverse-transcribed into cDNA using random hexamers (Amersham Biosciences, Buckinghamshire, UK) and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA).
Random hexamer
Manufactured by Cytiva
Sourced in United Kingdom, United States
Random hexamers are short, synthetic DNA sequences composed of six random nucleotides. They are commonly used in molecular biology applications as primers for complementary DNA (cDNA) synthesis during reverse transcription.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Moloney murine leukemia virus reverse transcriptase, by Promega (2 mentions)
Abi 7700 instrument, by Thermo Fisher Scientific (2 mentions)
Improm 2 reverse transcriptase, by Promega (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
User bulletin no 2, by Thermo Fisher Scientific (1 mentions)
Gene expression assay, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Sv total rna isolation system, by Promega (1 mentions)
Rnanow, by Ozyme (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Amv rt, by Promega (1 mentions)
Dnase digest, by Promega (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
19 protocols using random hexamer
1
MCAO-Induced Brain Tissue Analysis
2
Quantitative Analysis of Transporter Gene Expression
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Total RNA was isolated from cell lines and tumors using the SV Total RNA Isolation System (Promega, USA). A total of 1 μg of RNA was reverse transcribed to cDNA following M-MLV Reverse Transcriptase (Invitrogen, USA) and random hexamers (Amersham Pharmacia, UK) for reverse transcription. Analysis of hCNT1, hCNT2, hCNT3, hENT1, hENT2 and GAPDH (internal control) mRNA levels were performed by RT-PCR using TaqMan Gene Expression Assays (Applied Biosystems, USA) as previously described [44 (link)]. The mRNA expression of hOCT1 was assessed using the commercial Gene Expression Assays (Applied Biosystems). Relative quantification of gene expression was assessed using the ΔΔCT method, as described in the TaqMan user's manual (User Bulletin no. 2; Applied Biosystems). Gene expression levels for each individual sample were normalized relative to the GAPDH gene. The amounts of mRNA were expressed as arbitrary units.
Absolute quantification of gene expression was performed by using DNA plasmids containing each of the analyzed transporters to construct standard curves based on serial dilutions of the plasmids. The standard curves allowed us to correlate CT values of the samples with the mRNA copy number of each gene per microgram of total RNA.
Absolute quantification of gene expression was performed by using DNA plasmids containing each of the analyzed transporters to construct standard curves based on serial dilutions of the plasmids. The standard curves allowed us to correlate CT values of the samples with the mRNA copy number of each gene per microgram of total RNA.
3
RNA Extraction and Reverse Transcription
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Total RNA was extracted using the method of Chomczynski and Sacchi, (1987 (link)) according to the manufacturer's recommendations (RNANow, Ozyme, Saint-Quentin en Yvelines, France). Concentration and quality of the extracted RNA were assessed by spectrophotometry from 230 to 280 nm, using a Nanodrop 1000 spectrophotometer (Nanodrop Technology, Wilmington, USA). The ratios 260/280 and 260/230 were between 1.8 and 2.2. The integrity of RNA was assessed by the migration of total RNA on a 1.5% agarose gel. Total RNA samples (5 μg) were subjected to DNase (DNAfree, Invitrogen) and then reverse transcripted using RNase H-MMLV reverse transcriptase (Superscript II, Invitrogen, Cergy Pontoise, France) and random hexamers (Amersham, Orsay, France).
4
Quantitative RT-PCR Analysis of Apoptosis Genes
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Cells treated with inhibitors were lysed with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) for total RNA isolation. Subsequently, RNA extract was treated with TURBO DNA-free™ Kit (Ambion, Carlsbad, USA) in order to get rid of DNA contamination. The treated samples were thereafter subjected to cDNA synthesis using random hexamers (Amersham) at 65°C for 10 min and then at 37°C for 1 h. Quantitative RT-PCR was performed for gene specific transcript using primers specific for mouse were: caspase-8-Fwd, 5′-GAGGATGCCCAGAAGCTA-3′; caspase-8-Rev, 5′-CCG CAGCTCTCTCACCAT-3; caspase-3-Fwd, 5′-AATGTCATCTCGCTC TTGGT-3′; caspase-3-Rev, 5′-GCTTAGAATCACAC ACAC-3′; BAX-Fwd, 5′-CACCAGCTCTGAACAGAT-3′; BAX-Rev, 5′-CCAGTTCATCTCCAATTCG-3′. The reactions were carried out in an ABI Prism 7500 sequence detection system (Applied Biosystems, CA, USA). The qRT-PCR results were analyzed using the iCycler Thermal Cycler Software (Applied Biosystems, CA, USA) and normalized with those from 18S rRNA internal control (Das et al., 2009 (link)).
5
Real-Time RT-PCR Quantification Protocol
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Real-time reverse transcription-PCR (RT-PCR) was performed with 1 µg of total RNA, and reverse transcription was carried out using random hexamers (Amersham) with ImPromII reverse transcriptase (Promega). Quantitative PCR (qPCR) was performed using SYBR green PCR master mix (Applied Biosystems) in a total volume of 20 µl. Reactions were carried out on an ABI 7700 instrument (Applied Biosystems) using the standard cycling parameters. Every sample was examined at least three times in duplicate, and the results were normalized to a housekeeping gene (crp).
6
RNA Isolation and IDH1/2 Analysis
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Tumor tissue was snap-frozen in liquid nitrogen. Total RNA was isolated using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol and then reverse transcribed into complementary DNA (cDNA) using random hexamers (Amersham Biosciences, Buckinghamshire, United Kingdom) and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI).
IDH1 and IDH2 analysis using specific designed primers for Sanger analysis.
7
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was isolated using TRI Reagent (Sigma) according to manufacturer’s protocol. RNeasy Mini Kit was used for RNA purity optimization. Total RNA concentration was determined using NanoDrop ND1000 spectrophotometer (Nyxor Biotech, Paris). 1 μg of total RNA was transcribed reversely with 2 μg of random hexamers (Amersham, Taipei, Taiwan) and Superscript III reverse transcriptase (Invitrogen, Thermo Fisher Scientific Inc., Grand Island, NY, USA) according to manufacturer’s instructions. DEPC-treated water was used to dilute cDNA 100 folds and stored at −20 °C. Real-time PCR was done using SYBR Green PCR Master Mix using inbuilt System Software (Applied Biosystems, Life Technologies, Grand Island, NY, USA), 200nM forward and reverse primers, and cDNA equivalent of 0.5ug RNA. The triplicate PCR reaction conditions were as follows: 25 °C–5 min, 42 °C–60 min, 70 °C–5 min; total 45 cycles of 70 °C–10 min. 20ul PCR product was loaded to 1.5 % SYBR Green agarose gel for electrophoresis and checked under UV light. Gene expression was normalised to GAPDH and altered expression measured relative to the control (MDA-MB-231 Vector; shKDM5B-vector infected MDA-MB-231 cells).
8
Quantitative Analysis of Gene Expression
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Total RNA of cultured cells and human cardiac biopsies was isolated using TRIzol reagent (Invitrogen) followed by DNase digest (Promega) and ethanol precipitation according to standard protocols. Reverse transcription reactions were carried out via AMV-RT (Promega) with random hexamers (Amersham Pharmacia Biotech). Quantitative real-time PCR measurements were performed using the GoTaq qPCR Master Mix (Promega) and the ABI PRISM 7900HT Sequence Detection System. Gene expression was calculated using the ΔCT method with normalization to the housekeeping gene HPRT or GAPDH. Primer sequences are given in Supplementary Table S3.
9
Bacterial RNA Extraction and RT-PCR
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RNA from 500 µl of a bacterial culture was stabilized using the RNA Protect reagent (Qiagen) according to the manufacturer’s instructions. Total RNA was isolated using the RNeasy minikit (Qiagen) for total RNA isolation, and a RNase-free DNase set column (Qiagen) was used to eliminate DNA contamination. For real-time PCR (RT-PCR) experiments, 1 µg of total RNA was reverse transcribed using random hexamers (Amersham) by ImPromII reverse transcriptase (Promega). PCRs were performed using a 500 nM concentration of each gene-specific primer in a 10-µl volume with 1× SYBR green PCR master mix (Applied Biosystems). Reactions were run on an ABI 7700 instrument (Applied Biosystems) using the standard cycling parameters. Relative gene expression data analysis was carried out by the ΔΔCT method.
10
Gene Expression Analysis of Metabolic Regulators
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Total RNA was isolated from the tissue using Qiazol lysis reagent (Quiagen Sciences, USA). Two μg of RNA were treated with DNase I (DNase I amplification grade; Invitrogen, CA) and then reversely transcribed using random hexamers (Amersham Biosciences, NJ) and SuperScript II reverse transcriptase (Invitrogen, CA). Quantitative real-time polymerase chain reactions were performed using ABI PRISM 7900 System with TaqMan primers bmp7 (Mm00432102_m1), ppargc1a (Mm01208835_m1), fgf21 (Mm00840165_g1), elovl3 (Mm00468164_m1), rps29 (Mm02342448_gH), tbp (Mm01277042_m1), cidea (Mm00432554_m1), car4 (Mm00483021_m1), tfam (Mm00447485_m1), nrf1 (Mm01135606_m1) and reagents (Applied Biosystems, CA); Qiagen SYBR green primers were Prdm16 (QT00148127), tbx15 (QT00148127), zic1 (QT00173502), tcf21 (QT00100688), ucp1 (QT00097300), cpt1 (QT00172564) and tbp (QT00198443). Each reaction was performed in duplicate and results were normalized to the geometric average of two internal controls (Rps29 and Tbp).
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