RNA was isolated from cells by Trizol extractions (Invitrogen). Following treatment with DNase (Promega), two micrograms of total RNA was reversed transcribed with MultiScribe™ MuLV reverse transcriptase (Applied Biosystems). cDNA equivalent to 40 ng was used for quantitative polymerase chain reaction (qPCR) amplification (Applied Biosystems) with SYBR green PCR master mix (Applied Biosystems). Samples in which no reverse transcriptase was added (no RT) were included for each RNA sample. qPCR data were calculated using the comparative Ct method (Applied Biosystems). Standard deviations from the mean of the ΔCt values were calculated from three independent RNA samples. Primers are described in Supplemental Table 1. Where possible, intron spanning primers were used. All quantitative PCR was performed in triplicate, and three independent RNA samples were assayed for each time point. For measurements of relative gene expression, a fold change was calculated for each sample pair and then normalized to the fold change observed at HPRT and/or 18S rRNA.
Multiscribe mulv
Manufactured by Thermo Fisher Scientific
Sourced in Canada, United States
The MultiScribe™ MuLV is a reverse transcriptase enzyme used for the conversion of RNA to complementary DNA (cDNA). It is a high-performance, thermostable reverse transcriptase derived from the Moloney Murine Leukemia Virus (MuLV).
Automatically generated - may contain errors
Lab products found in correlation
Comparative ct method, by Thermo Fisher Scientific (3 mentions)
Trizol extraction, by Thermo Fisher Scientific (3 mentions)
Dnase, by Promega (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Taqman real time pcr system, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Qiazol reagent, by Qiagen (1 mentions)
6 protocols using multiscribe mulv
1
RNA Isolation, Reverse Transcription, and qPCR Analysis
2
Quantifying Gene Expression in Rhodnius prolixus
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Total RNA was extracted from tissues of adult female R. prolixus using TRIzol reagent (Invitrogen by Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. cDNA samples were synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Mississauga, ON, Canada), using 1 µg of total RNA, random primers and 50 U of MultiScribe MuLV reverse transcriptase. qPCR assays were performed as previously described (14 (link)). Actin and Rp49 (60S ribosomal protein) housekeeping genes were used as reference genes to normalize the target gene expression. To assess the accuracy of cDNA product amplification, the dissociation curves were examined and found to have a single peak produced for each pair of primers (Supplementary Table 1
3
Quantitative RT-PCR Expression Analysis
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RNA was isolated from cells by Trizol extractions (Invitrogen). Following treatment with DNase (Promega), two micrograms of total RNA was reversed transcribed with MultiScribe™ MuLV reverse transcriptase (Applied Biosystems). cDNA equivalent to 40 ng was used for quantitative polymerase chain reaction (qPCR) amplification (Applied Biosystems) with SYBR green PCR master mix (Applied Biosystems). Samples in which no reverse transcriptase was added (no RT) were included for each RNA sample. qPCR data were calculated using the comparative Ct method (Applied Biosystems). Standard deviations from the mean of the [Δ] Ct values were calculated from three independent RNA samples. Primers are described in Supplemental Table 1. Where possible, intron spanning primers were used. All quantitative PCR was performed in triplicate, and three independent RNA samples were assayed for each time point. For measurements of relative gene expression, a fold change was calculated for each sample pair and then normalized to the fold change observed at HPRT and/or 18S rRNA.
4
Quantification of Target Gene Expression by qRT-PCR
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Total RNA was isolated from NHBE cells and first-strand cDNA was generated using MultiScribe™ MuLV reverse transcriptase (Applied Biosystems). The first-strand cDNA was used to quantitate the mRNA levels by TaqMan real-time PCR system (Applied Biosystems). The level of expression of eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was used as reference, and fold change of target genes was calculated by the ∆∆CT method78 (link).
5
Real-Time PCR Gene Expression Analysis
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Total RNA was extracted from treated cells using QIAzol reagent (Qiagen, NL) according to the manufacturer's instructions. A total amount of 2 μg of RNA were transcribed to cDNA using MultiScribe™ MuLV and random primers (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems, CA, US). Real-time PCR was performed in an ABI Prism 7900HT Fast System Sequence Detection System (Applied Biosystems, CA, US) equipped with the SDS software (version 2.4.1) using SYBR Green (iQ™ SYBR® Green supermix, Bio-Rad Laboratories, DE) and primers designed with Primer-BLAST software (National Center for Biotechnology Information, MD, USA; https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ), according to published cDNA [30 (link)] or genomic sequences (Table S1 ) and with melting temperatures ranging from 58 to 60°C. A 2-fold dilution series was prepared from pooled cDNA samples to evaluate primer efficiency (E = 10[−1/slope]) and specificity as described elsewhere [32 (link)]. The relative expression was determined by the E-ΔΔCt method after internal normalization to Ppia as housekeeping gene.
6
Quantitative Analysis of Gene Expression
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RNA was isolated from cells by Trizol extractions (Invitrogen). Following treatment with DNase (Promega), two micrograms of total RNA was reverse transcribed with MultiScribe™ MuLV reverse transcriptase (Applied Biosystems). cDNA equivalent to 40 ng was used for quantitative polymerase chain reaction (qPCR) amplification (Applied Biosystems) with SYBR green PCR master mix (Applied Biosystems). Samples in which no reverse transcriptase was added (no RT) were included for each RNA sample. qPCR data were calculated using the comparative Ct method (Applied Biosystems). Standard deviations from the mean of the [Δ] Ct values were calculated from three independent RNA samples. Primers are described in Additional file 7 : Table S1. Where possible, intron-spanning primers were used. All quantitative PCR was performed in three technical replicates and three independent RNA samples representing biological replicates were assayed for each time point. For measurements of relative gene expression, a fold change was calculated for each sample pair and then normalized to the fold change observed at HPRT and/or 18S rRNA.
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