Venous blood was drawn into citrate buffer at the time of patient admission. Total RNA was extracted following manufacturer's instructions from 50 to 200 μl packed RBCs lysed in 1,300 μl Trizol® RNA preserving reagent (Invitrogen) stored at −80°C. Prior to cDNA synthesis, RNA was treated with DNase I (Sigma) to eliminate any contaminating genomic DNA. The sample was considered DNA free when fluorescence was still baseline after 30 cycles of quantitative PCR (qPCR) with seryl‐tRNA synthetase primers. cDNA was synthesized by reverse transcription using SuperScript® II and random hexamers (Invitrogen) at 25°C for 10 min, 42°C for 50 min and 70°C for 15 min. Genomic DNA was extracted from patient buffy coats by the automated Maxwell®16 system (Promega) or manually by the NucleoSpin® Blood kit (Macherey–Nagel). Although mainly patient DNA, sufficient amounts of P. falciparum DNA was extracted for the PCR amplification of relevant genes.
Superscript 2 and random hexamers
Manufactured by Thermo Fisher Scientific
SuperScript II is a reverse transcriptase enzyme used for the synthesis of first-strand cDNA from RNA templates. Random hexamers are short DNA oligonucleotides used as primers for reverse transcription reactions to generate cDNA from a wide range of RNA species.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq instrument, by Illumina (2 mentions)
Megaclear column, by Thermo Fisher Scientific (2 mentions)
Agilent hs dna chip, by Agilent Technologies (2 mentions)
Dnase treatment, by Thermo Fisher Scientific (2 mentions)
Microbexpress, by Thermo Fisher Scientific (2 mentions)
Absolute qpcr sybr green rox mix, by Thermo Fisher Scientific (2 mentions)
Rnaseh and e coli dna polymerase, by New England Biolabs (2 mentions)
Dnase 1, by Merck Group (1 mentions)
Nucleospin blood kit, by Macherey-Nagel (1 mentions)
Maxwell 16 system, by Promega (1 mentions)
Abi prism 7900 ht detection system, by Thermo Fisher Scientific (1 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using superscript 2 and random hexamers
1
RNA Extraction and cDNA Synthesis from Venous Blood
2
Microbial Community DNA and mRNA Sequencing
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Methods for microbial community DNA and mRNA sequencing were as previously described 44 (link),45 (link). Fecal pellets collected from mice before or after LPS treatment (pooled from 2 and 3 days post-injection) were kept at −80°C until DNA and RNA isolation using the guanidinium thiocyanate/cesium chloride gradient method 46 (link) as described, except that crude particles were removed by centrifugation prior to overlaying the gradient. RNA was subjected to DNase treatment (Ambion), purification using MEGAClear columns (Ambion), and rRNA depletion via subtractive hybridization (MICROBExpress, Ambion, in addition to custom depletion oligos). The presence of genomic DNA contamination was assessed by PCR with universal 16S rDNA primers. cDNA was synthesized using SuperScript II and random hexamers (Invitrogen, Carlsbad, CA), followed by second strand synthesis with RNaseH and E.coli DNA polymerase (New England Biolabs). Samples were prepared for sequencing with an Illumina HiSeq instrument after enzymatic fragmentation (NEBE6040L/M0348S). Libraries were quantified by quantitative PCR (qPCR) according to the Illumina protocol. qPCR assays were run using ABsoluteTM QPCR SYBR Green ROX Mix (Thermo Scientific) on an Mx3000P QPCR System instrument (Stratagene, La Jolla, CA). The size distribution of each library was quantified on an Agilent HS-DNA chip (Agilent, Santa Clara, CA).
3
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized using Superscript II and random hexamers (Invitrogen) or using the “RevertAidTM H Minus First Strand cDNA Synthesis Kit” (Thermo Fisher Scientific). Gene expression was quantified by SYBR Green-based qRT-PCR assays on the “Step one plus system” or by the ABI Prism HT 7900 Detection System instrument and software (Applied Biosystems). Data were analyzed by using the GraphPad Prism software tool (San Diego, CA, USA) or by the standard curve method for relative quantification, respectively. The primers for amplification of target transcripts are shown in Supplementary Table S3 .
All primer pairs were intron-flanking, except of the primers for 18S and MYOD. Amplification of 18S rRNA or GAPDH mRNA served to normalize the amount of sample cDNA. Gene expression analyses summarize at least two independent experiments performed as duplicates and measured in triplicates. Graphs represent the mean value of all measurements plus SEM.
All primer pairs were intron-flanking, except of the primers for 18S and MYOD. Amplification of 18S rRNA or GAPDH mRNA served to normalize the amount of sample cDNA. Gene expression analyses summarize at least two independent experiments performed as duplicates and measured in triplicates. Graphs represent the mean value of all measurements plus SEM.
4
Microbial Community DNA and mRNA Sequencing
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