Gene expression studies were performed on corpus callosum tissue isolated from five healthy and five animals fed 0.25% cuprizone in the standard rodent chow for 3 weeks, followed by 17 days on normal chow, and on spinal cord tissue isolated from four healthy and three EAE mice on day 17 post EAE induction. Total RNA was extracted using peqGOLD TriFast (VWR), and RNA concentration and purity were measured with the NanoDrop 1000 device (Thermo Scientific). cDNA synthesis was performed using moloney murine leukemia virus reserve transcriptase kit and random hexanucleotide primers (Thermo Fisher Scientific). cDNA levels were then analyzed by RT‐rtPCR using AceQ® qPCR SYBR Green Master Mix (Vazyme). The expression levels were calculated relative to the reference gene coding for hypoxanthin‐guanin‐phosphoribosyl‐transferase using the ΔΔCt method. Primer sequences and individual annealing temperatures are shown in Table 1 .
Random hexanucleotide primers
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Random hexanucleotide primers are short, synthetic DNA sequences that are commonly used in various molecular biology applications. They serve as primers for the initiation of DNA synthesis during processes such as reverse transcription and random priming. These primers have a random sequence of six nucleotides, which allows them to hybridize to multiple sites on a target DNA or RNA molecule, enabling the amplification or detection of genetic material.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (9 mentions)
Jumpstart taq dna polymerase, by Merck Group (8 mentions)
Sybr green, by Thermo Fisher Scientific (5 mentions)
Superscript 2 rnase h reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Icycler iq5 thermal cycler, by Bio-Rad (4 mentions)
Sybr green 1, by Thermo Fisher Scientific (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Truseq stranded mrna kit, by Illumina (2 mentions)
Sybr green nucleic acid stain, by Thermo Fisher Scientific (2 mentions)
Peqgold rna trifast, by Avantor (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Peqgold trifast, by Avantor (1 mentions)
Nanodrop 1000 device, by Thermo Fisher Scientific (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Gene specific primers, by GenePharma (1 mentions)
Lightcycler 480 thermal cycler, by Roche (1 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (1 mentions)
24 protocols using random hexanucleotide primers
1
Gene Expression Analysis in Demyelination and EAE
2
Quantification of Gene Expression
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A total of 3×105 cells/well were seeded into 6-well plates in triplicate and incubated at 37°C for 24 h. The cells were lysed, and total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific) according to the manufacturer's instructions. First strand cDNA synthesis was performed using reverse transcriptase and random hexanucleotide primers (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The complementary DNA was subsequently used to perform qPCR on a LightCycler480 Thermal Cycler (Roche Diagnostics, Basel, Switzerland) using SYBR Green (Molecular Probes; Thermo Fisher Scientific) with gene-specific primers (Gene Pharma, Shanghai, China) and JumpStart™ Taq DNA polymerase (Invitrogen; Thermo Fisher Scientific). The crossing threshold value was normalized to GAPDH, and quantitative changes in mRNA were expressed as fold-change relative to the control ± standard error of the mean (SEM) value.
3
RNA Extraction and Library Preparation for NGS
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Clarified infectious cell culture supernatant was filtered using 0.22-μm filters (Merck Millipore Co., MA, USA) to remove possible cellular residues. RNA was extracted using QIAamp viral RNA minikit (Qiagen, Hilden Germany) following the manufacturer’s protocol. RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and Qubit RNA 2.0 fluorometer using the Qubit RNA HS assay kit (Invitrogen, USA). Libraries for sequencing were prepared using TruSeq stranded mRNA kit (Illumina, USA), following the manufacturer’s protocol with the modification to exclude the poly(A)-containing mRNA purification steps. Reverse transcription was done using Superscript III reverse transcriptase (Invitrogen, USA) and random hexanucleotide primers (Invitrogen, USA). This was followed by second-strand synthesis using DNA polymerase I and RNase H, provided with the library preparation kit. Purification was performed using AMPure XP beads (Beckman Coulter, USA) after which the purified double-strand cDNA fragments were end repaired by adding a single A’ nucleotides to the 3′ end of the blunt fragments. Ligation of the adapters was performed, and the products purified and enriched by PCR to create the final library. Libraries were normalized, pooled, and sequenced using the Illumina platform.
4
RNA Extraction and Reverse Transcription
5
Robust DNA-Free RNA Isolation and cDNA Synthesis
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DNA-free RNA was obtained by treating 20 μL of nucleic acid solution with 4 U of DNase I (Invitrogen, Cergy Pontoise, France) in 1× DNase I reaction buffer at room temperature. DNA digestion repeated to remove remaining DNA traces, the reaction was stopped in presence of 1 μL of 25 mM EDTA (10 min at 65 °C), and RNA was purified with RNeasy Mini kit (Qiagen) according to manufacturer’s protocol. DNA contamination after the DNase I treatment of RNA samples was indicated by lack of qPCR amplification (performed as described below), and in the few cases where amplification did take place, an additional DNase I treatment was performed and no qPCR amplification took place then.
Total cDNA synthesis was carried out with 8 μL of resulting purified RNA extract, using random hexanucleotide primers (Invitrogen) and Omniscript reverse transcription kit (Qiagen) following the manufacturer’s instructions (90 min at 37 °C). The reverse transcriptase was inactivated 10 min at 95 °C, and cDNA was stored at − 20 °C.
Total cDNA synthesis was carried out with 8 μL of resulting purified RNA extract, using random hexanucleotide primers (Invitrogen) and Omniscript reverse transcription kit (Qiagen) following the manufacturer’s instructions (90 min at 37 °C). The reverse transcriptase was inactivated 10 min at 95 °C, and cDNA was stored at − 20 °C.
6
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA was extracted from cultured cells at 80% confluence using TRIzol® (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA synthesis was carried out using SuperScript II Reverse Transcriptase and random hexanucleotide primers (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. qPCR was performed using synthesized primers (Tsingke Biological Technology) and SYBR green master mix (Tiangen Biotech Co., Ltd.) to detect the mRNA levels. PCR conditions were as follows: Pre-denaturation at 95°C for 1 min; followed by 40 cycles of denaturation at 95°C for 20 sec, annealing at 60°C for 20 sec and elongation at 72°C for 30 sec. The reaction was performed using an Applied Biosystems 7500 Fast Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression levels of the target genes were quantitated using the 2−ΔΔCq method and β actin (ACTB) was used as the internal control to normalize the qPCR data (28 (link)). The primer sequences are presented in Table II . All samples were examined at least three times.
7
Transcriptome Sequencing Library Preparation
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Libraries for sequencing were prepared using TruSeq stranded mRNA kit (Illumina, USA), following the manufacturer’s recommended protocol with modification to exclude the poly(A)-containing mRNA purification steps. Briefly, reverse transcription on ∼25 ng/μl of RNA was achieved by using Superscript III reverse transcriptase (Invitrogen, USA) and random hexanucleotide primers (Invitrogen, USA). This was followed by second-strand synthesis using DNA polymerase I and RNase H, provided with the library preparation kit. Purification was then performed using AMPure XP beads (Beckman Coulter, USA) after which the purified double-strand cDNA fragments were end repaired by adding a single A nucleotide to the 3′ end of the blunt fragments, to prevent the formation of chimeras and improve adapter ligation efficiency. Ligation of the adapters was performed, and the products were purified and enriched by PCR to create the final library. Libraries were normalized and pooled before loading. Sequencing was carried out using the MiSeq reagent kit V3 (Illumina, USA), in a 600-cycle sequencing format.
8
cDNA Synthesis from Total RNA
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Total cellular RNA was extracted by TRIzol reagent (Life Technologies). RNA was eluted with RNase-free water, quantified by spectrophotometry and used for first-strand complementary DNA (cDNA) synthesis according to the manufacturer’s protocol (Applied Biosystems). Three micrograms of RNA was reversely transcribed to single-stranded cDNA. The reverse transcription was performed in a total volume of 50 μL containing 0.2 mM of each dNTP (Amersham Pharmacia Biotech, Piscataway, NJ), 10 μM of random hexanucleotide primers (Invitrogen), 200 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT) (Promega, Madison, WI), and 25 U RNAsin (Promega) at 37 °C for 2 h. The obtained cDNA was stored at −80 °C.
9
LRRK2-IN-1 Impacts AsPC-1 Cell Transcription
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AsPC-1 cells (105/well) were seeded into 6-well plates in triplicate in the presence of LRRK2-IN-1 and incubated at 37° C for 8 h. The cells were lysed, and total RNA was isolated using Tri Reagent (MRC) per the manufacturer’s instructions. First strand cDNA synthesis was carried out using SuperScript II Reverse Transcriptase and random hexanucleotide primers (Invitrogen). The complementary DNA was subsequently used to perform RT-PCR on an iCycler IQ5 Thermal Cycler (BioRad) using SYBR Green (Molecular Probes) with gene-specific primers and JumpStart™ Taq DNA polymerase (Sigma). The crossing threshold value assessed was normalized to β-actin and quantitative changes in mRNA were expressed as fold-change relative to control ± SEM value. The Student’s t-test was used to determine statistical significance. The primer sequences for the genes analyzed are provided in Additional file
1 : Table S1.
10
Quantitative Real-Time RT-PCR Analysis
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Total RNA was isolated from cells using TRIzol reagent (Invitrogen, CA, USA) and subjected to RT with Superscript II RNase H—reverse transcriptase and random hexanucleotide primers (Invitrogen). The cDNA was subsequently used for real-time RT-PCR by SYBR chemistry (SYBR Green I; Molecular Probes, Eugene, OR, USA) using gene-specific primers (Supplementary Table 1 ) and Jumpstart Taq DNA polymerase (Sigma-Aldrich). The crossing threshold value determined by real-time RT-PCR was noted for the transcripts and normalized with β-actin or U6 pri-miRNA. The changes in mRNA were expressed as fold change relative to control with±S.D. value.
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