RNA immunoprecipitation was performed following the Abcam protocol. Briefly, cells were cross-linked with formaldehyde (0.75%) and harvested after washing with 1 × PBS. Nuclei were pelleted by centrifugation and resuspended in RIP buffer [150 mM KCl, 25 mM Tris, pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 100 U/mL RNase inhibitor, and proteinase/phosphatase inhibitor cocktail (GenDEPOT)]. Chromatin was sheared by sonication and pelleted by centrifugation. For each IP, 5 μg of KSRP antibody was added, and the samples were incubated overnight at 4 °C with rotation. Protein A/G–agarose beads (Thermo) were added to the samples, which were rotated at 4 °C for 2 h, and then washed three times for 2 min each with RIP buffer. The RNA pellet precipitated with the beads was extracted directly with Trizol-LS (Invitrogen) and resuspended in 20 μl of RNase-free water. Total RNA, including KSRP-binding microRNAs, were reverse-transcribed using a microRNA reverse transcription kit (Qiagen). Mature miR-124 expression was quantified by quantitative PCR (qPCR) using a microRNA primer assay kit (Qiagen). U6 transcript was used as an internal control to normalize RNA input.
Microrna reverse transcription kit
Manufactured by Qiagen
Sourced in Germany, China
The MicroRNA Reverse Transcription Kit is a laboratory equipment product designed for the reverse transcription of microRNA (miRNA) into complementary DNA (cDNA). The kit provides the necessary reagents and protocols to facilitate the conversion of miRNA into cDNA, which is a crucial step in the analysis and quantification of miRNA expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Superscript reverse transcriptase kit, by Vazyme (2 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
Protein a g agarose beads, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr qpcr mix, by Toyobo (1 mentions)
Lightcycler, by Roche (1 mentions)
Rnase r, by Geneseed (1 mentions)
Thermal cycler cfx6 system, by Bio-Rad (1 mentions)
Quantitect sybr green rt pcr kit, by Qiagen (1 mentions)
Rnase r, by Illumina (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Sybr green real time pcr master mix, by Qiagen (1 mentions)
Abi prism 7900 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Mirneasy rna isolation kit, by Qiagen (1 mentions)
Lc480 system, by Roche (1 mentions)
Sybr green pcr master mix kit, by Vazyme (1 mentions)
7 protocols using microrna reverse transcription kit
1
Quantifying KSRP-Bound miR-124 via RIP-qPCR
2
Quantification of circRNA, mRNA, and miRNA
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Total RNA was isolated using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the user’s guideline. For mRNA and lncRNA quantification, complementary DNA was synthesized using SuperScript Reverse Transcriptase Kit (Vazyme, Nanjing, China). The transcript levels of circ_0047921 and LARP1 were evaluated by performing RT-qPCR assay on Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) based on the 2−ΔΔCt method, with β-actin as internal control. For miRNA quantification, microRNA Reverse Transcription Kit (Qiagen, Hilden, Germany) was used for reverse transcription, and U6 served as an internal control. Three wells were set for one sample in one experiment, and a total of three independent experiments were performed.
The primers were synthesized by Sangon Biotech (Shanghai, China) and sequences of primers were listed as follows:
The primers were synthesized by Sangon Biotech (Shanghai, China) and sequences of primers were listed as follows:
circ_0047921 (F, 5′-GCAGGCCAGCGCAGGA-3′; R, 5′-CAGGTTCCGAAACAATGT-3′);
miR-1287-5p (F, 5′-GCCGAGTGCTGGATCAGTGG-3′; R, 5′-CTCAACTGGTGTCGTGGA-3′);
LARP1 (F, 5′-GCTGTTTAGGAACAGCTGCC-3′; R, 5′-CCACAGGTGACAGGGAGAAG-3′);
β-actin (F, 5′-GCCGGGACCTGACTGACTAC-3′; R, 5′-TCTCCTTAATGTCACGCACGAT-3′); U6 (F, 5′-AACGCTTCACGAATTTGCGT-3′; R, 5′-CTCGCTTCGGCAGCACA-3′).
3
Circular RNA Expression Analysis
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Total RNA was prepared using Trizol reagent (Takara, Dalian, China) as recommended by the manufacturers. The extracted total RNA was reverse-transcribed into complementary DNA by reverse transcription kit (Takara) or microRNA reverse transcription kit (Qiagen, Hilden, Germany). Expression levels of RNAs were detected by SYBR® qPCR Mix (Toyobo, Tokyo, Japan) under the Roche Light-Cycler (Roche, Basel, Switzerland) based on the 2−ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and RNA U6 were used as endogenous controls. In addition, part of total RNA was exposed to RNase R (3 U/mg; Geneseed, Guangzhou, China) for 30 min at 37 °C to assess the stability of circ_0020850 and NUP98. The primer sequences were showed: circ_0020850-forward 5′-TAAAGATCGCCTGGCTCAGT-3′ and circ_0020850-reverse 5′-GGTTGTAGCCTGGCCAAAT-3′; NUP98-forward 5′-CTCCACCACTAATTCAGGCTTT-3′ and NUP98-reverse 5′-GAGGCTGGTAGTCTGCTGATT-3′; miR-326-forward 5′-GCCGAGCCTCTGGGCCCTTC-3′ and miR-326-reverse 5′-CAGTGCAGGGTCCGAGGTAT-3′; BECN1-forward 5′-GGTGTCTCTCGCAGATTCATC-3′ and BECN1-reverse 5′-TCAGTCTTCGGCTGAGGTTCT-3′; U6-forward 5′-GTGCTCGCTTCGGCAGCACA-3′ and U6-reverse 5′-GGAACGCTTCACGAATTTG-3′; GAPDH-forward 5′-CATCCATGACAACTTTGGTA-3′ and GAPDH-reverse 5′-CGTTGGCAGTGGGGACACGG-3′.
4
Circular RNA FUT8 Quantification Protocol
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Trizol reagent (Sigma) was applied to isolate the total RNA following the manufacturer’s protocols. The complementary DNA was acquired using a reverse transcription kit (Takara, Dalian, China) or microRNA reverse transcription kit (Qiagen, Hilden, Germany). To compare expression levels between groups, RT-qPCR was performed with a QuantiTect SYBR Green RT-PCR Kit (Qiagen) on thermal Cycler CFX6 System (Bio-Rad). The RT-qPCR results were analyzed by 2−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endogenous small nuclear RNA U6 as reference. Furthermore, PARIS™ Kit was purchased from Abcam for subcellular localization of circFUT8 according to the manufacturer’s instructions. The RNase R was purchased from Epicentre Technologies (Madison, WI, USA) for RNase R treatment assay. Total RNA was treated with or without 3U/mg RNase R for 15 min at 37°C, following by RT-qPCR assay.
The sequences of primers:
circFUT8 (sense-5ʹ-CACTCTAGCCGAGAACTGTCC-3ʹ; antisense-5ʹ-TTGTCCTGTACTTCATGCGCT-3ʹ);
linearFUT8 (sense-5ʹ-AACTGGTTCAGCGGAGAATAAC-3ʹ; antisense-5ʹ-TGAGATTCCAAGATGAGTGTTCG-3ʹ);
miR-944 (sense-5ʹ-GCCGAGAAATTATTGTACAT-3ʹ; antisense-5ʹ-CTCAACTGGTGTCGTGGA-3ʹ);
GAPDH (sense-5ʹ-CATCCATGACAACTTTGGTA-3ʹ; antisense-5ʹ-CGTTGGCAGTGGGGACACGG-3ʹ);
U6 (sense-5ʹ-CTCGCTTCGGCAGCACA-3ʹ; antisense-5ʹ-AACGCTTCACGAATTTGCGT-3ʹ).
The sequences of primers:
circFUT8 (sense-5ʹ-CACTCTAGCCGAGAACTGTCC-3ʹ; antisense-5ʹ-TTGTCCTGTACTTCATGCGCT-3ʹ);
linearFUT8 (sense-5ʹ-AACTGGTTCAGCGGAGAATAAC-3ʹ; antisense-5ʹ-TGAGATTCCAAGATGAGTGTTCG-3ʹ);
miR-944 (sense-5ʹ-GCCGAGAAATTATTGTACAT-3ʹ; antisense-5ʹ-CTCAACTGGTGTCGTGGA-3ʹ);
GAPDH (sense-5ʹ-CATCCATGACAACTTTGGTA-3ʹ; antisense-5ʹ-CGTTGGCAGTGGGGACACGG-3ʹ);
U6 (sense-5ʹ-CTCGCTTCGGCAGCACA-3ʹ; antisense-5ʹ-AACGCTTCACGAATTTGCGT-3ʹ).
5
Quantifying circHIPK3, HIPK3, miR-876-5p, and PIK3R1 in GC
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RNA samples were isolated from GC tissues and cells using Trizol reagent (Takara, Dalian, China) in accordance with the manufacturer’s instructions. The complementary DNA was synthesized using 2 μg of RNA template as template and reverse transcription kit (Takara) or microRNA Reverse Transcription Kit (Qiagen, Hilden, Germany). All complementary DNA products were quantitatively analyzed using SYBR Green Real-Time PCR Master Mix (Qiagen) under ABI Prism 7900 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The transcription levels of circHIPK3, HIPK3, miR-876-5p, and PIK3R1 were computed based on the 2−ΔΔCt method. Notable, phosphate dehydrogenase (GAPDH) and small nuclear RNA U6 were used to as housekeeping genes.
The sequences of primers were listed:
circHIPK3 (F-5′-GGGTCGGCCAGTCATGTATC-3′; R-5′-ACACAACTGCTTGGCTCTACT-3′);
HIPK3 (F-5′-TCACAAGTCTTGGTCTACCCA-3′; R-5′-CACATAGGTCCGTGGATAGTTTC-3′);
miR-876-5p (F-5′-GCCGAGTGGATTTCTTTGTG-3′; R-5′-CTCAACTGGTGTCGTGGA-3′);
PIK3R1 (F-5′-ACCACTACCGGAATGAATCTCT-3′; R-5′-GGGATGTGCGGGTATATTCTTC-3′);
GAPDH (F-5′-TCCCATCACCATCTTCCAGG-3′; R-5′-GATGACCCTTTTGGCTCCC-3′);
U6 (F-5′-AACGCTTCACGAATTTGCGT-3′; R-5′-CTCGCTTCGGCAGCACA-3′).
The sequences of primers were listed:
circHIPK3 (F-5′-GGGTCGGCCAGTCATGTATC-3′; R-5′-ACACAACTGCTTGGCTCTACT-3′);
HIPK3 (F-5′-TCACAAGTCTTGGTCTACCCA-3′; R-5′-CACATAGGTCCGTGGATAGTTTC-3′);
miR-876-5p (F-5′-GCCGAGTGGATTTCTTTGTG-3′; R-5′-CTCAACTGGTGTCGTGGA-3′);
PIK3R1 (F-5′-ACCACTACCGGAATGAATCTCT-3′; R-5′-GGGATGTGCGGGTATATTCTTC-3′);
GAPDH (F-5′-TCCCATCACCATCTTCCAGG-3′; R-5′-GATGACCCTTTTGGCTCCC-3′);
U6 (F-5′-AACGCTTCACGAATTTGCGT-3′; R-5′-CTCGCTTCGGCAGCACA-3′).
6
Quantifying miR-708 Expression via qPCR
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Small RNAs were extracted using the miRNEasy RNA Isolation Kit (Qiagen, Germantown, MD, USA), and RT reactions were performed using a TaqMan® MicroRNA Reverse Transcription Kit and TaqMan® MicroRNA assays specific to the mature form of miR-708. Real-time PCR reactions were performed using the TaqMan Universal PCR Master Mix No AmpErase UNG (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).
7
RNA Extraction and RT-qPCR Analysis
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TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) was applied to isolate total RNA from tissue samples or cells as instructed by the manufacturer. Subsequently, 5 μg of the RNA was reverse transcribed to complementary DNA with SuperScript Reverse Transcriptase Kit (Vazyme, Nanjing, China) and microRNA Reverse Transcription Kit (Qiagen, Hilden, Germany). RT-qPCR was performed to assess the relative expression level of RNA using SYBR Green PCR Master Mix kit (Vazyme) on Roche LC480 system (Roche Applied Science, Mannheim, Germany). The relative expression was examined using 2−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endogenous small nuclear RNA U6 as the internal control.
The specific primers used were:
The specific primers used were:
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