Thirty male or twenty female flies from each of five independent repeats were collected and submerged in 1.4 ml of RNAlater reagent (Thermo Fisher Scientific). Total RNA was isolated using the RNeasy kit (QIAGEN Inc.) in accordance with the manufacturer’s protocol. mRNA was reverse transcribed using the RevertAid Premium reverse transcriptase (Thermo Fisher Scientific). Briefly, 5 mM of oligo(dT), 1 mM of deoxyribonucleotide triphosphates and 5 μg of total RNA were mixed in a PCR tube to a total volume of 10 μl, incubated for 5 min at 65 °C and stored on ice. In a second tube, 200 units (U) of RevertAid Premium reverse transcriptase were mixed with 1 × reverse transcriptase buffer and 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific) in a total volume of 10 μl. Finally, the volume in the two tubes was combined and incubated in a thermocycler for 5 min at 50 °C, 5 min at 85 °C, 1 s at 22 °C and stored on ice. The 2 × Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific) was mixed with extra ROX Reference dye to a final concentration of 0.5 μM. Per well of a 96-well plate, the cDNA samples to be analysed were diluted 1:75 or 1:150 and mixed with 1 × SYBR Green/ROX Reference Dye and 0.25 μM of the forward and reverse primers.
Revertaid premium reverse transcriptase
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
RevertAid Premium Reverse Transcriptase is a recombinant, modified M-MuLV reverse transcriptase enzyme used for the synthesis of first-strand cDNA from RNA templates. It exhibits high thermal stability and enhanced priming efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (7 mentions)
Lightcycler 480 real time pcr system, by Roche (4 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Lightcycler 480 sybr green 1 master, by Roche (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Random primer, by Thermo Fisher Scientific (3 mentions)
Oligo dt primer, by Thermo Fisher Scientific (3 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Qbase 2, by CellCarta (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Rnasin plus rnase inhibitor, by Promega (2 mentions)
Steponeplus real time pcr instrument, by Thermo Fisher Scientific (2 mentions)
Qiazol, by Qiagen (2 mentions)
Maxima sybr green rox master mix, by Thermo Fisher Scientific (2 mentions)
Maxima sybr green rox qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
67 protocols using revertaid premium reverse transcriptase
1
Fly RNA Isolation and Reverse Transcription
2
Quantification of GFP mRNA in Transformed Bacteria
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was isolated from bacterial cells transformed with pET28b-EGFP containing different inserts as described (Masulis et al., 2015 (link)). Ten micrograms of RNA and 2 pmol of a 32P labeled gfp-specific primer 5′-CTCTGGTCAGGCAGATACCTCTGGTCAG-3′ were used for the synthesis of cDNA by RevertAid Premium reverse transcriptase (Thermo Fisher Scientific, United States). Samples were treated with RNase A (Thermo Fisher Scientific; 10 U, 37°C, 30 min), precipitated with threefold volume of 96% ethanol and 0.3M sodium acetate and washed with 70% ethanol. The precipitate was dissolved in 5 μl of 98% formamide with 8 mM NaOH and 4 mM EDTA, fractionated in 8% polyacrylamide gel (PAAG) with 8M urea and radioautographed.
3
Quantification of Oxidative Stress Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using a Macherel-Nagel NucleoSpin RNA Kit according to the manufacturer’s instructions. cDNAs were generated from 2 μg of total RNA using random hexamers and RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific). The following primers were used for the TaqMan® probe-based qPCR assay (Applied Biosystems): DUOX1 (Hs00213694_m1), DUOX2 (Hs00204187_m1), NOX4 (Hs00276431_m1), NOX5 (Hs00225846_m1), RELA (Hs00153294_m1), SEPP1 (Hs01032845_m1), CATALASE (Hs00156308_m1), GPX1 (Hs00829989_gH), SOD2 (Hs00167309_m1), TXNRD1 (Hs00917067_m1), NQO1 (Hs01045993_g1), FTL (Hs00830226_gH), MGST1 (Hs00220393_m1) DUOX1 (Hs00213694_m1), and DUOX2 (Hs00204187_m1). Beta-ACTIN (Hs99999903_m1) served as the internal control. The sequences of the primers used for the SYBR assays are shown in Supplemental Data Table S15 . Quantitative RT–PCR was performed using the Applied Biosystems 7300 Real-Time PCR System. All experiments were performed in triplicate.
4
Gene Expression Analysis of PCD Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of marker genes involved in PCD regulation was measured with real time quantitative PCR (qPCR). Three biological repeats were used for gene expression analysis with qPCR. RNA was treated with DNAseI and reverse transcription was performed using 2 μg of RNA with the RevertAid Premium Reverse Transcriptase (RT) and Ribolock Rnase inhibitor according to manufacturer’s instructions (Thermo Fisher Scientific). After reverse transcription the reaction was diluted to the final volume of 100 μl. 1 μl was used for PCR with EvaGreen ROX (Solis Biodyne). The cycle conditions in the ABI 7900HT Fast RT PCR System (Applied Biosystems) were: 95°C 10 min, 40 cycles with 95°C 15 s, 60°C 30 s, 72°C 30 s and ending with melting curve analysis. Normalization of the data was performed in qBase 2.0 (Biogazelle), with three reference genes TIP41, YLS8 and SAND. Primer amplification efficiencies were determined in qBase from a cDNA dilution series. Primer sequences and amplification efficiencies can be found in S1 Table . Data normality was tested and subsequently 2-base logarithmed for statistical analyses. Factorial ANOVA posthoc analyses Fisher LSD was used to evaluate significant differences between mutant and leaf age, and One-Way ANOVA to changes in gene expression with leaf age (Statistica 7.1, Stat Soft Inc).
5
Quantifying mRNA Expression by qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA isolation and qRT-PCR analysis were performed as previously described [54 (link)]. Briefly, total RNA was isolated using the TRIzol Total RNA Isolation kit (Sangon Biotech, Shanghai, China) and treated with DNase I (Sangon Biotech, Shanghai, China). Eight hundred nanograms of total RNA was reverse-transcribed using RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and diluted ten-fold for PCR amplification. The PCR was performed on a LightCycler480 II instrument (Roche, Basel, Switzerland). Each reaction contained 2 μL of cDNA template, 10 μL of SYBR Green qPCR Master Mix (BBI, Toronto, ON, Canada), and 0.2 μmol L−1 gene-specific primers in a final volume of 20 μL. The PCR conditions included an initial incubation at 95 °C for 3 min, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. The specificity of the PCR reactions was determined by melting curve analyses of the products. Relative expression levels were calculated by the 2−ΔΔCT method. The rice Actin1 gene was used as the internal control. The primer sequences are listed in Table S9 .
6
Quantification of miRNAs and mRNA in Hippocampus and Cortex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from hippocampal and cortical tissues using the SanPrep Column microRNA Extraction kit (Sangon Biotech Co., Ltd.), and cDNA was synthesized by reverse transcription using Revert Aid Premium Reverse Transcriptase (Thermo Fisher Scientific, Inc.). Subsequently, 2X SG Fast qPCR Master Mix (Sangon Biotech Co., Ltd.) was used to amplify the 10 candidate miRNAs. The PCR thermocycling conditions of the 10 candidate miRNAs were as follows: 95°C for 3 min, followed by 35 cycles at 94°C for 30 sec, 57°C for 30 sec, and 72°C for 30 sec, ending at 72°C for 8 min. The miRNA expression levels were normalized to those of U6 small nuclear RNA. Copy numbers of the 10 miRNAs were obtained using the standard curve of RT-PCR. To detect Nrn1 mRNA levels, total RNA was isolated from cortical and hippocampal tissue samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Equivalent amounts of RNA from each sample were used for cDNA synthesis using the FastKing gDNA Dispelling RT SuperMix kit [Tiangen Biotech (Beijing) Co., Ltd.]. Subsequently, the cDNAs were used for qPCR analysis. The thermocycling conditions of Nrn1 mRNA are 95°C for 15 min, followed by 40 cycles at 95°C for 10 sec, 60°C for 20 sec, and 72°C for 30 sec. The primer sequences are listed in Table I and the 2−ΔΔCq method was used to analyze the level of mRNA (22 (link)).
7
Bilberry Fruit RNA Extraction and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from bilberry fruits at stage 2 that were collected after 0, 6, 12, 24 and 48 h from the beginning of the light treatments. The RNA was isolated according to the method of Jaakola et al. [38 (link)] with the exception that the phenol-chloroform extraction was substituted with the RNA purification protocol in E.Z.N.A.® Total RNA Kit I (Omega Bio-Tek, Norcross, GA, USA). The quality of the isolated RNA was verified by measuring the absorbance spectrum with NanoDrop N-1000 spectrophotometer (NanoDrop Technologies, Thermo Scientific, Wilmington, DE, USA) and on a 1% (w/v) ethidium bromide-stained agarose gel. RNA was converted to cDNA with RevertAid Premium Reverse Transcriptase (Thermo Scientific) in accordance with the manufacturer’s instruction. RNA extraction (and further gene expression analyses) was repeated twice for each set of plants.
8
Bacterial RNA Extraction and qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To collect RNA, bacteria were harvested from liquid culture by centrifugation at different time points. The cells were mechanically disrupted in TRI Reagent (Sigma) using glass beads. The subsequent steps were performed using a GeneJET RNA Purification Kit (Thermo Scientific). DNA contamination was removed with an On-Column DNase I Digestion Set (Sigma). The reverse transcription reaction was carried out according to the manufacturer’s protocol using RevertAid Premium Reverse Transcriptase (Thermo Scientific), 2 μg of total RNA as the template, and random hexanucleotides (Genomed). The qPCR reaction mixture was composed of 5 μl of the reverse transcription samples diluted 100-fold in water, 0.75 μl of a pair of gene-specific primers (10 μM each; Supplementary Materials Table S1 ), 1 μl of nuclease-free water, and 7.5 μl of iTaq Universal SYBR Green Super Mix (Bio-Rad). The reactions were carried out in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) according to the following program: 1. 95 °C 2 min; 2. 95 °C 30 s; 3. 55 °C 30 s (fluorescence measurement); 4. 74 °C 30 s; 5. repeat steps 2–4 40 times; melting curve analysis from 65 to 95 °C with a 0.5 °C increase step. The data obtained were analysed using the ΔΔCt method53 (link). The 23S rRNA was used as a reference.
9
RNA Extraction and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected protoplasts were collected 48h post-transfection by centrifugation at 80 x g, immediately frozen in liquid nitrogen and stored at –80°C. Plant samples were collected at 17 dpi, immediately frozen in liquid nitrogen and stored at –80°C. Frozen tissue were ground to a fine powder using a Retsch Mixer Mill MM400 and ~ 60 mg of material was used for RNA extraction. Total RNA extraction was performed as previously described [90 (link)] using Trireagent (MRC), and the concentration and purity of the RNA samples was determined using a Nanodrop spectrophotometer. Following quantification, all RNA samples were normalized to 200 ng/μl, and 500 ng of total RNAs were then treated with DNase I (ThermoFisher) and reverse-transcribed using random hexamer primers (5 μM) and Revertaid Premium Reverse Transcriptase or Maxima H-minus Reverse Transcriptase (ThermoFisher) in the presence of Ribolock RNase inhibitor (ThermoFisher) according to the manufacturer's instructions. cDNA synthesis was confirmed by PCR amplification of EFIα cDNA using gene-specific primers (Table 3 ), and verification by agarose gel electrophoresis for the presence of a product of appropriate size.
10
Isolation and Quantification of RNA from Drosophila Larval Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Third instar larval brains dissected in Schneider medium were homogenized in 150 µl immunoprecipitation (IP) buffer [50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, 10% glycerol, complete EDTA-free protease inhibitor and RNase inhibitor (RNAsin Plus RNase Inhibitor, Promega)]. The lysate was precleared with 45 µl of washed Pierce Control Agarose Resin (Thermo Fisher Scientific). For each reaction, 50 µl of pre-cleared lysate was taken as a 50% input sample directly to RNA extraction. Next, 100 µl of pre-cleared lysate was incubated with 25 µl of GFP-TRAP agarose beads (Chromotek) for 2 h at 4°C with rotation. The beads were washed four times briefly each with 200 µl cold IP buffer at 4°C. After the final wash, beads were re-suspended in 100 µl extraction buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA and 1.3% SDS, 1:100 RNAsin) and incubated at 65°C, 1000 rpm (mixing frequency) for 30 min in a thermomixer. The elution step was repeated and the supernatants were pooled. RNA was extracted from inputs and immunoprecipitates with the RNAspin RNA Isolation kit (GE Healthcare) and eluted in 40 µl of nuclease-free H2O. Reverse transcription was performed using RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific) with random hexamer primers. cDNA was then used as a template for real-time quantitative PCR.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!