TRIzol reagent (Sigma, USA) was used for total RNA isolation. MicroRNA reverse transcription kit (Takara, Dalian, China) was used to reverse transcribe cDNA. RT-qPCR assay was performed by using SYBR Green Master Mix II (Takara). U6 and GAPDH are used as internal references. The relative expression of miR-448 and ROCK1 was calculated by the 2−△△ct method. The primer sequences have been shown in Table 1 .
Microrna reverse transcription kit
Manufactured by Takara Bio
Sourced in Japan, China, United States
The MicroRNA Reverse Transcription Kit is a laboratory tool designed to convert microRNA molecules into complementary DNA (cDNA) for further analysis. The kit provides the necessary reagents and protocols to efficiently perform reverse transcription of microRNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Sybr green master mix 2, by Takara Bio (2 mentions)
Trizol reagent, by Merck Group (2 mentions)
Primescript rt kit, by Takara Bio (2 mentions)
Sybr green pcr master mix, by Toyobo (2 mentions)
Abi 7500 fast pcr system, by Toyobo (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
High capacity rna to cdna kit, by Takara Bio (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Takara Bio (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
Cdna synthesis kit, by Bio-Rad (1 mentions)
Mircute mirna qpcr detection kit, by Tiangen Biotech (1 mentions)
Trizol reagent, by Beyotime (1 mentions)
Beyofast sybr green one step qrt pcr kit, by Beyotime (1 mentions)
Universal genomic dna purification mini spin kit, by Beyotime (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Gdna eraser, by Takara Bio (1 mentions)
Abi 7500 qrt pcr system, by Thermo Fisher Scientific (1 mentions)
18 protocols using microrna reverse transcription kit
1
Quantitative Analysis of miR-448 and ROCK1
2
Quantification of lncRNA and miRNA Expression
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TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was adopted to extract lncRNAs and miRNAs out of BV2 microglia and HT-22 neurons. The PrimeScript® RT reagent kit (TaKaRa, Dalian, Liaoning, China) and the MicroRNA Reverse Transcription kit (Takara, Shiga, Japan) were employed to reverse transcribe the lncRNAs and miRNAs into cDNAs, respectively. Next, real-time fluorescence quantitative PCR was implemented with the use of SYBR Green PCR reagent and ABI7500FAST Real-Time PCR. The 2-ΔΔCt method was introduced to assess the profiles of MIR497HG and miR-29b-3p. GAPDH was taken as the internal parameter of MIR497HG, and U6 was regarded as that of miR-29b-3p. MIR497HG: forward: 5′-ATAAGAATCCAGGTCGGGGC-3′, reverse: 5′-CCCAAGGTTCCATCGTCCTC-3′; miR-29b-3p: forward: 5′-GGGGGTACCCTTCAGGAAGCTGGTTTC-3′, reverse: 5′-GGGGATATCTACATGTGAGGCAGGTTCTCAC-3′; GAPDH: forward: 5′-GAAGATGGTGATGGGATTTC-3′, reverse: 5′-GAAGGTGAAGGTCGGAGT-3′; U6: forward: 5′-CTCGCTTCGGCAGCACA-3′, reverse: 5′-AACGCTTCACGAATTTGCGT-3′.
3
Quantification of miRNA Expression
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Total RNA was isolated using TRIzol reagent (Invitrogen, USA). Reverse transcription was performed using the microRNA reverse transcription kit (Takara Bio, Otsu, Japan). RT‐qPCR was performed in a 10 μl reaction system containing forward/reverse primers, complementary DNA and SYBR Green MasterMix (Takara Bio) with three replicates. The miRNA levels were normalized to the endogenous control U6 small nuclear RNA. The relative expression of these RNAs was calculated using the 2−∆∆CT method. The primers used in the study are listed in Table 1 .
4
Quantification of miRNA Expression
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Total RNA was prepared with the Axygen® AxyPrep Multisource RNA Miniprep Kit (Corning, USA) and cDNA was transcribed with the cDNA Synthesis Kits (Bio-Rad, USA) or the MicroRNA Reverse Transcription Kit (Takara, Japan). The SYBR® Green Master Mix (Bio-rad, USA) or miRcute miRNA qPCR Detection Kit was used for the qRT-PCR (TIANGEN, China). For miR-1-3p, miR-4443, and miR-516a-5p, internal reference was U6. The primers were produced by Sangon Biotechnology Inc. (Shanghai, China). The sequence of primers was as follows:
U6 (Forward : CTCGCTTCGGCAGCACA, Reverse : AACGCTTCACGAATTTGCGT), hsa-miR-1-3p (Forward : CGGGCTGGAATGTAAAGAAG, Reverse : CAGCCACAAAAGAGCACAAT), hsa-miR-4443 (Forward : CGGGCTTGGAGGCGT, Reverse : CAGCCACAAAAGAGCACAAT), hsa-miR-516a-5p (Forward : CGGGCTTCTCGAGGAAAGAAG, Reverse : CAGCCACAAAAGAGCACAAT), hsa-miR-561-5p (Forward : CGGGCATCAAGGATCTTAAA, Reverse : CAGCCACAAAAGAGCACAAT).
U6 (Forward : CTCGCTTCGGCAGCACA, Reverse : AACGCTTCACGAATTTGCGT), hsa-miR-1-3p (Forward : CGGGCTGGAATGTAAAGAAG, Reverse : CAGCCACAAAAGAGCACAAT), hsa-miR-4443 (Forward : CGGGCTTGGAGGCGT, Reverse : CAGCCACAAAAGAGCACAAT), hsa-miR-516a-5p (Forward : CGGGCTTCTCGAGGAAAGAAG, Reverse : CAGCCACAAAAGAGCACAAT), hsa-miR-561-5p (Forward : CGGGCATCAAGGATCTTAAA, Reverse : CAGCCACAAAAGAGCACAAT).
5
Comprehensive miRNA and circRNA Expression Analysis
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Total RNA in this study was isolated from cultured cells and tissues using Trizol reagent (Invitrogen, USA). Then, extracted RNAs were reverse-transcribed into cDNA by using Prime Script™ RT kit (Takara, Dalian, China). In addition, for miRNAs, MicroRNA Reverse Transcription Kit (Takara Biotechnology, Japan) were used to perform reverse transcription. The primers used in the current experiments were deigned and purchased from Geneseed Biomart (Guangzhou, China) (Table 1 ). qRT-PCR was carried out on ABI 7500 fast PCR System (Carlsbad, CA, USA) with a SYBR green PCR Master Mix (TOYOBO, Japan). GAPDH and U6 applied as internal references for mRNAs and miRNAs, respectively. The relative expressions were calculated with 2–ΔΔCT method. For RNase R treatment, 1 unit of RNase R was added to digest 1 μg of RNA for 15 min at 37 °C.
Primers used in the present study.
| Gene | Primer sequence |
|---|---|
| miR-187-3p | forward: 5′-CACAGGACCCGGGCG-3′ |
| reverse: 5′-CCGGCTGCAACACAAGAC-3′ | |
| miR-665 | forward: 5′-CTCGCTTCGGCAGCACA-3′ |
| reverse: 5′-CAGTGCGTGTCGTGGAGT-3′ | |
| U6 | forward: 5′-CTCGCTTCGGCAGCACA-3′ |
| reverse: 5′-AACGCTTCACGAATTTGCG′ | |
| hsa_circRNA_104348 | forward: 5′-TCTGTGTGTCAAAGCAAGGC-3′ |
| reverse: 5′-AGATGCCACTGAATCACCCA-3′ | |
| RTKN2 | forward: 5′-CTCATGGTGTGCAATGCTCG-3′ |
| reverse:5′-ACTGCAGATAGAGAAATGGACTT-3′ | |
| GAPDH | forward: 5′-CTCTGCTCCTCCTGTTCGAC-3′reverse: 5′- GCGCCCAATACGACCAAATC-3′ |
6
Gene Expression Analysis via qRT-PCR
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TRIzol reagent (Beyotime, Shanghai, China) was used to extract total RNA. BeyoFast™ SYBR Green One-Step qRT‒PCR Kit (Beyotime) was used to detect targeting gene according to the manual. In addition, for miRNAs, MicroRNA Reverse Transcription Kit (Takara Biotechnology, Japan) were used to perform reverse transcription. qRT-PCR was carried out on ABI 7500 fast PCR System (Carlsbad, CA, USA) with a SYBR green PCR Master Mix (TOYOBO, Japan). β-actin and U6 applied as internal references for mRNAs and miRNAs. The relative expressions were calculated with 2–ΔΔCT method. Moreover, genomic DNA (gDNA) was extracted by a Universal Genomic DNA Purification Mini Spin Kit (Beyotime) based on the manufacturer’s protocol. All primers were synthesized by GenePharma (Shanghai, China) and are listed in Table S2 .
7
Quantitative RT-PCR Analysis of mRNA and miRNA
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The total RNA of the L4-L6 spinal cord section was extracted using TRIzol reagent (Takara Bio., Japan) following the manufacturer’s instructions. Complementary DNA (cDNA) was then synthesized through reverse transcription of the RNA using the Prime-Script RT reagent kit and gDNA Eraser (Takara) or MicroRNA Reverse Transcription Kit (Takara). Subsequently, a quantitative reverse transcription polymerase chain reaction (qRT-PCR) of the DNA transcript was then performed using the SYBR PremixEx TaqII Kit (Takara) in the ABI 7500 qRT-PCR system (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6 were used as the internal control. The expression levels of mRNA and miRNA were assessed based on the 2-ΔΔCt equation. The primers (Sangon Biotech, Shanghai, China) used in this study are shown in Table 1 .
8
Quantitative Analysis of SNHG15, miR-490-3p, and HDAC2
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Total RNA isolation was performed with TRIzol reagent (Sigma, United States). The cDNA was reverse transcribed using microRNA reverse transcription kit (TAKARA, Dalian, China). RT-qPCR assay was performing using SYBR Green Master Mix II (TAKARA). U6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference for RNA or protein. Relative expression of SNHG15, miR-490-3p and HDAC2 were detected with the 2-ΔΔct method.
9
Quantification of miRNA and mRNA Expression
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Total cellular RNA was extracted from the cultured melanoma cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Reverse transcription was performed using 1.0 μg total RNA and a MicroRNA Reverse Transcription Kit (Takara Biotechnology, Japan) or Prime Script™ RT kit (Takara, Dalian, China), which were used to investigate the expression of miRNA and mRNA, respectively. Amplification reactions was carried out on an ABI 7500 Sequencing Detection System (Applied Biosystems, CA, USA) with a SYBR green PCR Master Mix (Takara, Japan). All reactions were run in triplicate and were normalized to the miRNA house-keeping gene U6 or the mRNA house-keeping gene GAPDH. All primers are listed in Table 1 .
10
Quantitative RNA Expression Analysis
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Total RNA from NSCs, BMSCs, and exosomes with different transfections or treatments was isolated using TRIzol reagent (Takara, Dalian, China). The cDNA Reverse Transcription Kit (Takara) or MicroRNA Reverse Transcription Kit (Takara) was used to reverse transcribe 450 ng of RNA. Quantitative real-time PCR analysis was performed with Applied Biosystems 7500 real time PCR system (Thermal Fisher, Waltham, USA) using TB Green Premix Ex Taq II (Takara, Dalian, China). The reaction conditions were: 40 cycles, 95°C for 5s, and 60°C for 34 s. The specific primers used were as follows:
miR-199a-5p (F: 5′-CCCAGUGUUCAGACUACCUGUUC-3′),
miR-26a-5p (F: 5′-GCCGGTTCAAGTAATCCAG-3′),
miR-185-3p (F: 5′-CACTCCAGCTGGGTTTCCTCTGGTCC-3′),
miR-6314 (F: 5′-CAGGCACTGACAGATCTGATGGT-3′),
U6 (F: 5′-CTCGCTTCGGCAGCACA-3′; R: 5′-AACGCTTCACGAATTTGCGT-3′),
GSK-3β (F: 5′-CACAGAACCTCTTGCTGGAT-3′; R: 5′-GGTGCCCTGTAGTACCGAGA-3′), and
GAPDH (F: 5′-GTCGGTGTGAACGGATTTG-3′; R: 5′-TCCCATTCTCAGCCTTGAC-3′). The expression of miRNAs were normalized to the expression of U6 and calculated by using the 2
−ΔΔCT method.
miR-199a-5p (F: 5′-CCCAGUGUUCAGACUACCUGUUC-3′),
miR-26a-5p (F: 5′-GCCGGTTCAAGTAATCCAG-3′),
miR-185-3p (F: 5′-CACTCCAGCTGGGTTTCCTCTGGTCC-3′),
miR-6314 (F: 5′-CAGGCACTGACAGATCTGATGGT-3′),
U6 (F: 5′-CTCGCTTCGGCAGCACA-3′; R: 5′-AACGCTTCACGAATTTGCGT-3′),
GSK-3β (F: 5′-CACAGAACCTCTTGCTGGAT-3′; R: 5′-GGTGCCCTGTAGTACCGAGA-3′), and
GAPDH (F: 5′-GTCGGTGTGAACGGATTTG-3′; R: 5′-TCCCATTCTCAGCCTTGAC-3′). The expression of miRNAs were normalized to the expression of U6 and calculated by using the 2
−ΔΔCT method.
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