Total RNA was isolated using TRIzol method (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The first-strand complementary DNA (cDNA) was synthesized for gene expression analysis and miRNA expression using random primer (Roche, Basel, Switzerland) and miR-130a-specific reverse transcription primer (RiboBio Co., Ltd., Guangzhou, China), respectively. The real-time RT-PCR for the quantification of HCV, IFNα, IFNβ, and GAPDH mRNAs was performed with the FastStart Universal SYBR Green Master Mix (Roche). All the assays were performed as described [12 (link)].
Random primer
Manufactured by Roche
Sourced in Switzerland, Germany, United States
Random primers are short, synthetic oligonucleotide sequences that are used as primers in various molecular biology applications. They are designed to randomly bind to multiple regions within a DNA or RNA template, allowing for the amplification of unknown or unpredictable sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Lightcycler 480, by Roche (7 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Transcriptor reverse transcriptase, by Roche (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
M mlv rt, by Promega (3 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Rnase free dnase set, by Qiagen (3 mentions)
Moloney murine leukemia virus reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 sybr green 1 master reaction mix, by Roche (2 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
Rnaseout, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
54 protocols using random primer
1
Quantification of HCV and Interferons
2
Laser-Microdissected Cell RNA Extraction
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The extraction of total RNAs from clinical samples of laser-microdissected cells was performed as described previously [8 (link)]. The amplified RNA or total RNA was reverse transcribed to generate single-stranded cDNAs using a random primer (Roche) or oligo(dT)16 primer with Superscript II reverse transcriptase (Roche). We prepared appropriate dilutions of each single-stranded cDNA for subsequent PCR amplification by monitoring tubulin, alpha 3 (TUBA3) as a quantitative control. The primer sequences were 5’-CTTGGGTCTGTAACAAAGCATTC-3’ and 5’-AAGGATTATGAGGAGGTTGGTGT-3’ for TUBA3 and 5’-GTCCTGAAAGTCAAGCACCTG-3’ and 5’-GAAGTTCTTGTTGGTGCTTATGG-3’ for C16orf74. All reactions involved initial denaturation at 94°C for 2 min, followed by 22 cycles (for TUBA3) or 28 cycles (for C16orf74) of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min on a GeneAmp PCR system 9700 (PE Applied Biosystems).
3
RNA Isolation and qRT-PCR Analysis
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The E.Z.N.A.® Total RNA Kit I (Catalogue Number R6834–01, Omega Bio-Tek, USA) was utilized to isolate RNA from cell lysates. From 1 µg of isolated RNA, a Transcriptor First Strand cDNA Synthesis Kit and Random Primer (Catalogue Number 04897030001, Roche, USA) were used to generate cDNA according to the manufacturer’s protocol. The 7500 Fast Real-Time PCR system (Applied Biosystems, USA) was utilized for qRT-PCR. A relative quantification method was used to analyse the qRT-PCR data, and actin was used as a reference for the mRNAs or lncRNAs. Each sample was analysed in triplicate. The primer sequences synthesized by Shanghai Generay Biotech Co.,Ltd, are listed in Table S3 .
4
RT-qPCR Analysis of Gene Expression
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RNA was isolated from tissues using the RNeasy kit (Qiagen) according to the manufactures instructions and total RNA from cultured cells was isolated using a Nucleospin RNA kit (Fisher Scientific). cDNA synthesis was performed using 1 μg of total RNA isolated from tissues or from cells and mastermix containing a blend of random primer (Roche), RT enzyme (Roche) and dNTP (Roche)and real-time PCR amplification was performed as described previously [13 (link)] using a LightCycler® 480 Real-Time PCR System (Roche, Dublin, Ireland). The results were normalised to the β actin gene and relative expression was calculated based on the 2-ΔΔCt method as previously described [17 (link)]. All gene-specific primer sequences used in the study are listed in S1 and S2 Tables.
5
Expression Analysis of VEGI, DR3, and DcR3
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Total RNA was extracted from the U-2 OS, SaOS-2 and HMVE cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription of 2 µg of total RNA was performed at 42˚C for 1 h using random primer (Roche Diagnostics, Tokyo, Japan) and reverse transcriptase (Roche Diagnostics), and the cDNAs thus produced were sequentially amplified by PCR with Takara Ex Taq™ DNA polymerase (Takara bio, Ohtsu, Shiga, Japan) using the following specific primer sets: sense, 5'-CTCTGCACTG GGAACATGAA-3' and antisense, 5'-TTGGCTCAGGGTA GCTGTCT-3' for VEGI; sense, 5'-CGCAGA GATACTGACTG TGG-3' and antisense, 5'-AGGAGGTGCTAGAAGGGTGT-3' for DR3; sense, 5'-GCTACTGCAACGTCCTCTG-3' and antisense, 5'-ACACTCCTCAGCTCCTGGTA-3' for DcR3; sense, 5'-GTCATCAATGGAAATCCCATCACC-3' and antisense, 5'-GCTCAGGGATGACCTTGCCC-3' for GAPDH. The amplification conditions for PCR of VEGI, DR3 and DcR3 included 35 cycles at 95˚C for 30 sec, 57˚C for 1 min and 72˚C for 1 min, followed by heating at 72˚C for 7 min, while that for GAPDH included 30 cycles at 95˚C for 30 sec, 57˚C for 30 sec and 72˚C for 1 min, followed by heating at 72˚C for 7 min. The amplified fragments were resolved by electrophoresis in 1.5% agarose gel and detected using ethidium bromide staining.
6
Reverse Transcription Protocol for cDNA Synthesis
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A reverse transcription kit was used to obtain the cDNA, specifically a transcriber First Strand cDNA synthesis kit and Random Primer of Roche, used according to the manufacturer's directions. The final microtube was taken to the conventional ThermoCycler (Labnet) using a protocol of 10-min at 25 ºC, 30-min at 55 ºC, 5-min at 85 ºC, and cooling at ∞ 4 ºC; finally, the cDNA was obtained and quantified in the Fluorometer Qubit® 2.0.
7
Osteogenic Gene Expression in MC3T3-E1 Cells
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Total RNA was isolated from the MC3T3-E1 pre-osteoblasts seeded at 1 × 105 cells/disc coated with OCP without/with κ-carrageenan after 1, 14, and 21 days of culture using an Invitrogen RNA isolation kit (Invitrogen). cDNA synthesis was performed using 0.5–1 μg total RNA in 20 μl reaction mix consisting of 5 units Transcriptor Reverse Transcriptases (Roche Diagnostics, Basel, Switzerland), 1 mM of each dNTP (Invitrogen), 0.08 A260 units random primers (Roche Diagnostics), and 1x concentrated Transcriptor Reverse Transcriptase reaction buffer (Roche Diagnostics). Real-time PCR (RT-PCR) reactions were performed using the LightCycler® 480 SYBR green I Master reaction mix according to the manufacturer’s instructions (Roche Diagnostics) in a Light Cycler 480 (Roche Diagnostics), and relative housekeeping gene expression (PBGD) and relative target gene expression, i.e. runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), fibroblast growth factor 2 (Fgf2), dentin matrix protein 1 (Dmp1), and osteopontin (Opn) were determined. Primers (Invitrogen) used for RT-PCR are listed in Table 1 . Values of target gene expression were normalized for PBGD gene expression.
8
RNA Extraction and RT-qPCR Analysis
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RNA was extracted using the RNeasy kit (Qiagen, Crawley, UK), according to the manufacturer’s instructions. For cDNA synthesis, 300–500 ng RNA was used in combination with 0.5 μg random primers (Roche, West Sussex, UK), 40 U RNaseOUT 0.5 μM dNTPs (Invitrogen, Paisley, UK) 1× RT Buffer (Fermentas, Cambridge, UK) and RevertAid reverse transcriptase (Fermentas, Cambridge, UK) made up to a final volume of 20 μl using RNase-free water (Qiagen, Crawley, UK). Reactions were carried out in a thermocycler with conditions—25 °C for 10 min, 42 °C for 60 min and 70 °C for 10 min. One microlitre cDNA per well on a 96-well plate (Roche) was used for RT-qPCR with SYBR Green reagent and remaining cDNA stored at −80 °C. RT-qPCRs were performed using a LightCycler 480 Instrument II (Roche, West Sussex, UK). Gene expression was normalised to Hprt and relative expression calculated by the ΔΔCT method [40 (link)]. Each RT-qPCR contained 1× buffer, 0.4 mM dNTPs, 50 μM primers (Additional file 1 : Table S1), 0.01 U Taq DNA polymerase (Invitrogen, Paisley, UK) and nuclease-free water (Qiagen, Crawley, UK). Four primer sets for Dnmt1, Dnmt3a, Dnmt3b [47 (link)] and Hprt [13 (link)] were used. The general thermocycler conditions are as follows—94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 63 °C for 1 min, 72 °C for 1 min with a final elongation step of 72 °C for 4 min.
9
Quantitative RNA Expression Analysis
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Brain, whole gastrocnemius muscle tissue, and isolated muscle fibers from gastrocnemius muscle were homogenized in Qiazol Lysis reagent (Qiagen, #79309) using a TissueLyser II (Qiagen) at 30 Hz. Qiazol lysates from tissues and cell lines were cleared by centrifugation at 12,000×g for 10 min at 4 °C and RNA extracted by chloroform. The aqueous phase was used for RNA isolation using QIAwave RNA mini kit (Qiagen, #74536) with on-column DNA digestion (Qiagen RNase-Free DNase Set cat #79256) according to the manufacturer's instructions. 2 μg RNA were transcribed to cDNA using random primers (Roche, #11034731001), dNTPs (Invitrogen, #10297018), RNaseOUT (Invitrogen, #10777-019) and SuperScript III Reverse Transcriptase (Invitrogen, #18080-044) according to the manufacturer's instructions. qPCR was performed using Taqman probes and TaqMan Fast Advanced Master mix (ThermoFisher, #4444556) on a Roche Lightcycler480. Gene expression was calculated using the delta–delta Ct method and normalized to the house keeping gene Rn18S. The Taqman probes used are described in the Supplementary Table 3 .
10
RNA Isolation, cDNA Synthesis, and qRT-PCR Analysis
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RNA isolation from the transfected cells from fresh or frozen plates was done using Trizol® reagent (Sigma, Marlborough, MA, USA) according to the manufacturer’s protocol. The conversion of RNA into cDNA was done using 1 μg of RNA, random primers (Roche, Basel, Switzerland), and M-MLV reverse transcriptase enzyme (Invitrogen, Darmstadt, Germany) according to the manufacturer’s protocol. Gene expression analysis was performed by real time polymerase chain reaction (RT-PCR) (Applied Biosystems® 7500 Real Time PCR, Applied Biosystems, Darmstadt, Germany) in standard conditions provided by the manufacturer employing Taqman® (Applied Biosystems, Thermo Fisher Scientific, Darmstadt, Germany) gene-specific probes for G6PD, NRF2, HMOX1, and NQO1. The expression levels were quantified using the ΔΔCt method using peptidylprolyl isomerase A (PPIA) as a reference gene.
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