Total RNA from cell lines and tissue samples was extracted using TRIzol (Invitrogen, USA) following the manufacturer’s instructions. For the plasma, the total RNAs were extracted using an mirVana PARISTM microRNA extraction kit (ABI, USA) following the manufacturer’s protocols. For lncRNA quantification, GAPDH was used as internal control, and PrimeScript RT Master Mix (QIAGEN, Germany) was used for reverse transcription and real-time polymerase chain reaction (PCR). For circRNA expression detection, RNase R was added to remove linear RNA after the sample RNA was extracted, and then random primers were reverse transcribed into cDNA for further amplification. For the design of circRNA amplification primers, this study adopts the design method of trans circularization site primers. The primer sequences of all genes are shown in Additional file 1 : Table S1. All reactions were performed in triplicate. The fold change for each gene relative to the control group was calculated using the 2−ΔΔCt method.
Primescript rt master mix
Manufactured by Qiagen
Sourced in Germany
PrimeScript RT Master Mix is a ready-to-use solution for reverse transcription (RT) reactions. It contains all the necessary components for efficient cDNA synthesis from RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Qiagen (1 mentions)
Pc707, by Astec (1 mentions)
Mx3000p qpcr system, by Agilent Technologies (1 mentions)
Premix ex taq, by Takara Bio (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
7 protocols using primescript rt master mix
1
Comprehensive RNA Extraction and Quantification
2
Exosome RNA Extraction and qRT-PCR Analysis
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The extraction of whole-RNA from exosome pellets, cells, or tissues was performed by TRIzol reagent (Invitrogen) according to the standard procedure. Then Prime Script RT Master Mix and SYBR Premix Ex Taq (Qiagen, Valencia, CA, USA) were utilized for cDNA generation and qRT-PCR analysis, respectively. The relative expression was assessed using 2−ΔΔCt method with the internal references of GADPH and U6. The sequence of primers was listed: circ_0008717: F 5’-GCCTTTCCATTCCGTCAGGA-3’, R 5’-ACCACGCTCAAAACAAAGGTG-3’; miR-1287-5p: F 5’-AGCTGGATCAGTGGTTCGAG-3’, R 5’-CAGTGCAGGGTCCGAGGTAT-3’; PAK2: F 5’-TCTTCCTCCCCCAGGGTTG-3’, R 5’-AATCGAGCCCACTGTTCTGG-3’; GAPDH: F 5’-GAAGGTGAAGGTCGGAGTC-3’, R 5’-GAAGATGGTGATGGGATTTC-3’; U6: F 5’-GCTTCGGCAGCACATATACTAAAAT-3’, and R 5’- CGCTTCACGAATTTGCGTGTCAT-3’.
3
Extracting and Analyzing Zebrafish Embryo RNA
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Total RNA of whole zebrafish embryos was extracted with QIAzol Lysis Reagent (Cat. No. 79306; Qiagen, Venlo, Netherlands), followed by the RT reaction using an PrimeScript RT Master Mix (Cat.
No. RR036A, Takara Bio Inc., Shiga, Japan). PCR was performed using a Taq PCR Core Kit (Ca. No. 201223; Qiagen) in a standard thermal cycler PC707 (ASTEC, Fukuoka, Japan) under the thermal cycling conditions of 94 °C for 30 s, 53 °C for 30 was blindly performed by densitometry using the ImageJ software and normalized to the levels of elfa.
No. RR036A, Takara Bio Inc., Shiga, Japan). PCR was performed using a Taq PCR Core Kit (Ca. No. 201223; Qiagen) in a standard thermal cycler PC707 (ASTEC, Fukuoka, Japan) under the thermal cycling conditions of 94 °C for 30 s, 53 °C for 30 was blindly performed by densitometry using the ImageJ software and normalized to the levels of elfa.
4
Quantifying DCTN1 Expression in Drosophila
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The expression of DCTN1 in neurons and other tissues in Drosophila has been reported previously [8 (link)]. Total RNA was isolated from adult flies (five flies per sample) using the RNeasy Lipid Tissue Mini Kit (Qiagen). cDNA was synthesized from total RNA using the PrimeScript RT Master Mix (Qiagen), and real-time PCR was performed using the Mx3000P qPCR System (Agilent) and Premix Ex Taq (Takara Bio). The primer sequences were as follows:
dDCTN1 forward: 5′-AGCCGTGCCAGGTTTG-3′
dDCTN1 reverse: 5′-CGTTCGCCCTCATACA-3′
rp49 forward: 5′-AGCGCACCAAGCACTTCATCCGCCA-3′
rp49 reverse: 5′-GCGCACGTTGTGCACCAGGAACTTC-3′
dDCTN1 forward: 5′-AGCCGTGCCAGGTTTG-3′
dDCTN1 reverse: 5′-CGTTCGCCCTCATACA-3′
rp49 forward: 5′-AGCGCACCAAGCACTTCATCCGCCA-3′
rp49 reverse: 5′-GCGCACGTTGTGCACCAGGAACTTC-3′
5
Ago-RIP Assay in hADSCs for circRNF111
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The Magna RNA Immunoprecipitation (RIP) Kit (cat. no., Millipore, Bedford, MA, United States) was employed to carry out the Ago-RIP assay in hADSCs cells as described by the manufacturer. About 1 × 107 differentiated hADSCs were sedimented, followed by re-suspension in an equivalent pellet volume of the RIP Lysis Buffer enriched with protease inhibitor cocktail along with RNase inhibitors. Thereafter, 200 μl of the cell lysate was inoculated with antibody against Ago2 (10 μg) (cat. no., Millipore, Billerica, MA, United States) or the control rabbit IgG-coated beads and incubated overnight at 4°C while mixing by rotation. Subsequently, the reaction mixture was inoculated with proteinase K buffer, and the RNeasy MinElute Cleanup Kit (cat. no., Qiagen, Düsseldorf, Germany) employed to isolate the immunoprecipitated RNAs, followed by generation of cDNA using the Prime- Script RT Master Mix (cat. no., TaKaRa, Tokyo, Japan). RT-qPCR was employed to assess circRNF111 relative abundance, with GAPDH serving as the internal control.
6
RNA Extraction and lncRNA Quantification
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Total RNA from cell lines and tissue samples was extracted using TRIzol (Invitrogen, USA) following the manufacturer's instructions. For the plasma, the total RNAs were extracted using an mirVana PARISTM microRNA extraction kit (ABI, USA) following the manufacturer's protocols. For lncRNA quanti cation, GAPDH was used as internal control, and PrimeScript RT Master Mix (QIAGEN, Germany) was used for reverse transcription and real-time polymerase chain reaction (PCR). The primer sequences are listed in Additional le 1: Table S2. All reactions were performed in triplicate. The fold change for each gene relative to the control group was calculated using the 2 -ΔΔCt method.
7
RNA Extraction and lncRNA Quantification
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Total RNA from cell lines and tissue samples was extracted using TRIzol (Invitrogen, USA) following the manufacturer's instructions. For the plasma, the total RNAs were extracted using an mirVana PARISTM microRNA extraction kit (ABI, USA) following the manufacturer's protocols. For lncRNA quanti cation, GAPDH was used as internal control, and PrimeScript RT Master Mix (QIAGEN, Germany) was used for reverse transcription and real-time polymerase chain reaction (PCR). The primer sequences are listed in Additional le 1: Table S2. All reactions were performed in triplicate. The fold change for each gene relative to the control group was calculated using the 2 -ΔΔCt method.
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