Cultured cells were harvested at various time points. Total RNA was extracted from cells using RNAiso Plus reagent (Takara, Dalian, China, http://www.takara.com.cn ). Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis System (Thermo Fisher Scientific) in a 20 µl reaction volume. After reverse transcription, 180 µl of water was added to the reaction mixture, and 1 µl of aliquot was used for each reaction. Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) was performed using Power SYBR Green PCR Master Mix (Life Technologies,) and StepOne Plus Real‐Time PCR system (Applied Biosystems, Life Technologies). β‐actin was used as an internal control. Primers (Supporting Information Table 2) were designed and generated by Sangon Biotech (Shanghai, China, http://sangon.bioon.com.cn/ ).
Revert aid first strand cdna synthesis system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Revert Aid First Strand cDNA Synthesis System is a kit for the synthesis of first-strand complementary DNA (cDNA) from RNA templates. It includes a thermostable reverse transcriptase, RNase inhibitor, and necessary buffers and reagents.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Takara Bio (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Quantitect sybr green kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Abi 7300, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Quantinova sybr green pcr kit, by Qiagen (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
6 protocols using revert aid first strand cdna synthesis system
1
Quantifying Gene Expression by qRT-PCR in Cultured Cells
2
Quantitative Analysis of TET Isoforms
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Total RNA was extracted using RNeasy Mini Kit (Qiagen), and reverse transcription was performed by RevertAid First Strand cDNA Synthesis system (ThermoScientific), followed by qRT-PCR using QuantiTect SYBR Green Kit (Qiagen). ACTB and GAPDH were used as internal references for normalization. Primers were not isoform specific and therefore measure expression of TET1 (TETFL and TET1ALT) and TET2 (isoform a,b) collectively. See Additional file 1 : Table S1 for primer sequences.
3
Gene Expression Analysis in Spinal Cord Transplants
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Seven days after cell transplantation, spinal cord tissues from the engrafted cortices were collected and homogenised on ice. Total RNA was extracted using TRIzol reagent (Takara Bio Inc., Otsu, Japan), and the concentration and purity were determined. Total RNA was reverse transcribed into complementary DNA using the Revert Aid First Strand cDNA Synthesis System (Invitrogen). The primer sequences information was as follows: NR_037671, sense, 5′-GTCACGGCTGAGTAAGAAT-3′, antisense, 5′-AACTATACCCAGGCACAAT-3’; Cntnap5a, sense, 5′-TGTTACTGAGGACAAGATTTGG-3′, antisense, 5′-TTCTCAGAGTTGGAACCCT-3’. The amplification was performed using a DNA thermal cycler (ABI 7300; Applied Biosystems, Waltham, MA, USA). β-actin was used as an internal control to evaluate the normalised ΔCt value of each sample. The thermocycling conditions were set as follows: initial denaturation at 94 °C for 5 min, and 35 cycles of amplification at 94 °C for 1 min and 72 °C for 1 min. The relative expression of the targeted gene was normalised to that of β-actin by the 2─ΔΔCt method.
4
Quantitative PCR Analysis of Gene Expression
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Cortical and hippocampal tissues were collected, and total RNA was extracted using RNAiso plus (Takara Bio Inc., Otsu, Japan). The concentration and purity of total RNA were determined using a microplate reader (BioTek, Shanghai, China), which was then reverse transcribed into complementary DNA using the Revert Aid™ First Strand cDNA Synthesis System (Invitrogen, Carlsbad, CA, USA). The primers were designed for the detected factors using Primer 5.0 software (Premier, San Francisco, CA, USA), as shown in Table 2
5
Quantitative PCR Analysis of Gene Expression
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The total RNA fraction was extracted using Trizol reagent (Takara Bio Inc., Otsu, Japan) and reverse transcribed using RevertAid™ First Strand cDNA Synthesis System (Invitrogen). Quantitative PCR reactions were carried out with Power SYBR (DBI Bioscience) according to the manufacturer’s instructions. The expression level of each gene was normalized to that of GAPDH using the 2−ΔΔct method. Primers used for the reactions are as follows:
GAPDH: forward: TGACTTCAACAGCGACAC CCA,
GAPDH: reverse: CACCCTGTTGCTGTAGC CAAA;
GAP43: forward: TGTT GCCGATGGGGTGGAGA,
GAP43: reverse: CCGTTGGAGGCTGGGCTGTT;
PTN: forward: CAGTGGAGTGTGTGTGTGCC;
PTN: reverse: GATTCTGCTTGAGGTT TGGG;
STAT3: forward: GACAAAGACTCTGGGGACG,
STAT3: reverse: ATTG GGGGCTTGGTAAAAA;
JAK2: forward: CGGCTGGGCAGTGGAGAGT,
JAK2: reverse: CGGTGATGGTGCGATTTGG.
GAPDH: forward: TGACTTCAACAGCGACAC CCA,
GAPDH: reverse: CACCCTGTTGCTGTAGC CAAA;
GAP43: forward: TGTT GCCGATGGGGTGGAGA,
GAP43: reverse: CCGTTGGAGGCTGGGCTGTT;
PTN: forward: CAGTGGAGTGTGTGTGTGCC;
PTN: reverse: GATTCTGCTTGAGGTT TGGG;
STAT3: forward: GACAAAGACTCTGGGGACG,
STAT3: reverse: ATTG GGGGCTTGGTAAAAA;
JAK2: forward: CGGCTGGGCAGTGGAGAGT,
JAK2: reverse: CGGTGATGGTGCGATTTGG.
6
Quantification of Gene Expression by RT-qPCR
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Total RNA of cells was extracted using Trizol reagent (15596018, Thermo Fisher Scientific), and the Nanodrop (Thermo Scientific, San Diego, CA, USA) was used to measure the concentration of RNA, and then the RNA concentration was diluted to 500 ng/μL. Total RNA (1 µg) was converted into cDNA using a RevertAid firststrand cDNA synthesis System (K1621, Invitrogen). The genome DNA was erased by genome DNA wiper kit in the procedure of cDNA synthesis. The mRNA expression levels were determined by SYBR-Green PCR Master Mix (4309155, Thermo Fisher Scientific) in the 7500 Real-Time PCR system (Thermo Fisher Scientific). The following components were combined in a 10 μL solution: 4 μL cDNA, 5 μL SYBR, 1 μL Primer. The PCR cycle was as follows: pretreatment at 95°C for 1 min for pre-denaturation, at 95°C for 30 sec, at 58°C for 20 sec, at 70°C for 20 sec (three steps all in 40 cycles) for amplification. The expression levels of RT-PCR products were determined by the 2 -ΔΔCT method (Livak and Schmittgen, 2001) . All primer sequences are listed in Table 1.
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