Total RNA from cells and tissues was extracted by using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from total RNA by using reverse transcription kit (Beyotime, China). The cDNAs were amplified with Power SYBR green PCR master mix (Beyotime, China), with 18 S rRNA or 36b4 as an endogenous control. The results were collected from QuantStudio 6 Flex by Thermo Fisher Scientific instrument. The qPCR was done in triplicate and repeated at least 3 times. Primer information for RT-qPCR is listed in Supplementary information.
Reverse transcription kit
Manufactured by Beyotime
Sourced in China
The Reverse Transcription Kit is a laboratory tool designed to convert RNA into complementary DNA (cDNA) through the process of reverse transcription. This kit provides the necessary reagents and enzymes to perform this essential step in various molecular biology applications, such as gene expression analysis, cDNA library construction, and RT-PCR.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Total rna extraction kit, by Takara Bio (2 mentions)
Sybr green qpcr supermix, by Thermo Fisher Scientific (2 mentions)
Quantstudio 6 flex, by Thermo Fisher Scientific (1 mentions)
Rnaeasy micro kit, by Qiagen (1 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Rt qpcr kit, by Takara Bio (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Trizol, by Beyotime (1 mentions)
Chemiluminescence imager, by Bio-Rad (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Exicycler 96, by Bioneer (1 mentions)
Total rna extraction kit, by Tiangen Biotech (1 mentions)
Sybr green qpcr mix, by Beyotime (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Miscript reverse transcription kit, by Qiagen (1 mentions)
Quantinova sybr green pcr kit, by Qiagen (1 mentions)
Abi 7500, by Thermo Fisher Scientific (1 mentions)
Rnaeasy kit, by Beyotime (1 mentions)
16 protocols using reverse transcription kit
1
RNA Extraction and qPCR Analysis
2
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted and cDNA was synthesized as described previously.10 RNA was isolated using the RNAeasy Micro Kit (Qiagen) according to the manufacturer's instructions. RNA concentration and quality were measured using a NanoDrop spectrophotometer (Thermo). cDNA templates were synthesized from RNA (2 μg) using a reverse transcription kit (Beyotime).
3
Stemness and Differentiation Gene Expression Analysis
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To analyze gene expression of stemness and differentiation, qRT-PCR was performed. 3D co-axial cells were extracted by dissociation solution as described above and 2D cell were also collected. Total RNA was isolated and extracted by total RNA extraction kit (Takara, Beijing, China). RNA was reversely transcribed into cDNA with reverse transcription kit (Beyotime Biotechnology, Shanghai, China) according to the protocol. For qPCR, pairing primer sequences [20 (link)] were used (Supplementary Table S1 ), including alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2), osteopontin (OPN), Runt-related transcription factor 2 (Runx-2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR amplification was performed using SYBR Green qPCR SuperMix (C11733046, Invitrogen, NY, USA) with an ABI PRISM® 7500 Sequence Detection System (ThermoFisher Scientific) with the procedure of 95°C 2 min; 95°C 15 s, 60°C 30 s, reading plate, 40cycles. Each sample was repeated three times.
4
Neuroblastoma Cell Line Cultivation
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Human neuroblastoma M17 cells [human neuroblastoma cell line BE(2)-M17; cat. no. CL-0262] were purchased from Shanghai Chunmai Biotechnology Co.; RPMI-1640 medium was purchased from Hyclone (GE Healthcare Life Sciences); reverse transcription kit from Beyotime Institute of Biotechnology; RT-qPCR kit from Takara Biotechnology Co., Ltd.; TRIzol kit from Shanghai Pufei Biotechnology Co., Ltd.; fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) from Sigma-Aldrich (Merck KGaA); UV-3100PC spectrophotometer from Shanghai MeiPuda Co., Ltd.; Lipofectamine 2000 transfection reagent from Invitrogen (Thermo Fisher Scientific, Inc.), and MTT detection kit from Thermo Fisher Scientific, Inc.
5
Quantitative Real-Time PCR Analysis
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In accordance with cultured 786-O, 769-P, and HK-2 cells, total RNA was obtained by TRIzol (Beyotime, Shanghai, China), and then reverse transcribed into cDNA by reverse transcription kit (Beyotime, Shanghai, China) on PCR Cycler (Bio-Rad, United States). We employed SYBR-Green mixture (Beyotime, Shanghai, China) and Bio-Rad chemiluminescence imager (Bio-Rad, United States) for qRT-PCR. All the above experimental steps were conducted in accordance with the product instructions, and the amplified primer sequences were as follows:
GAPDH served as internal control, the relative expression was examined by 2^−ΔΔCt. The experiment was repeated three times.
GAPDH served as internal control, the relative expression was examined by 2^−ΔΔCt. The experiment was repeated three times.
6
Quantifying GPR30 Expression in BCAS Mouse Model
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30 min, 3 h, 6 h, and 24 h after BCAS, the mice were anesthetized with isoflurane (5%) and decapitated. Thereafter, the hippocampi were isolated on ice. Total RNA was extracted with Trizol reagent (Takara Bio Inc.) and reversely transcription into cDNA using reverse transcription kit (Beyotime Institute of Biotechnology, China). Cycle parameters were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of sequential incubations at 95°C for 15 s and at 60°C for 1 min. After the end of the cycle, the data was analyzed using the instrument's supporting software to obtain the cycle threshold (Ct value). GPR30 expression was normalized to glyceraldehyde-3-phosphatedehydrogenase (GADPH) and calculated by the 2-∆∆CT method as previously described [31 (link)]. The primers were listed as follows: GPR30 (forward 5′-TCATTTCTGCCATGCACCCA-3′ and reverse 5′-GTGGACA-GGGTGTCTGATGT-3′) and GAPDH (forward 5′-AACTTTGGCATTGTGGAAGG-3′ and reverse 5′-ACACATTGGGGGTAGGAACA-3′).
7
Quantitative Gene Expression Analysis
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Four types of cell strains were used for total RNA was extraction. Reverse transcription was performed to convert the extracted RNA into cDNA using the reverse transcription kit provided by Beyotime, https://www.beyotime.com/ . Exicycler 96 (BIONEER) was used to detect its fluorescence expression quantity. All results were processed with GAPDH for standardization. The 2-ΔΔ Ct method was used to calculate the relative expression levels of genes.
8
Alzheimer's Disease Gene Expression Analysis
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To analyze gene expression levels of stemness, differentiation and specific proteins of Alzheimer's disease, qRT-PCR was conducted with several kits according to the manufacturer's instructions. The bioprinted 3D co-axial cells were extracted by dissociation solution as described above. Total RNA was isolated and extracted by total RNA extraction kit (Takara). The quantity and purity of RNA was assessed using a NanoDrop Spectrophotometer (Thermo Scientific). RNA was reversely transcribed into cDNA with reverse transcription kit (Beyotime Biotechnology) according to the protocol. For qRT-PCR, pairing primer sequences were used (Table S1 ), including Nestin, Tubulin (TUJ1), glial fibrillary acidic protein (GFAP), APP, 3-repeat and 4-repeat tau (3R4RTau), 3-repeat tau (3RTau), 4-repeat tau (4RTau) and β-actin (ACTB). PCR amplification was performed using SYBR Green qPCR SuperMix (C11733046, Invitrogen) with an ABI PRISM® 7500 Sequence Detection System with the procedure of 95 °C 2 min; 95 °C 15 s, 60 °C 30 s, reading plate, 40 cycles. Each sample was repeated 3 times. Relative gene expression was calculated using the 2−ΔΔCt method. The same procedures were performed for 2D and mixed control groups.
9
Quantifying mRNA Expressions via qRT-PCR
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To determine mRNA expressions, total RNA was extracted from cells by the total RNA extraction kit (DP419, TIANGEN, China). Subsequently, the complementary DNA from each sample was synthesized by reverse transcription reactions using a reverse transcription kit (D7166, Beyotime, China). The qRT-PCR was conducted by a real-time PCR system (7300, Applied Biosystems, USA) with SYBR Green qPCR Mix (D7260, Beyotime, China) as per the guide. The primers are given in Table 1 . The expression levels of circ_0001998-1 and miR-145 were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 expression by the 2−ΔΔCt method.
10
Quantification of Gene Expression
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The PCR primers for SNRPB, LSM10, ATXN2, PRPF3, EDC3, and β action were designed with NCBI primer Blast tool. Detailed primer sequences of these genes were listed in Supplement Table S1 . The total RNA was extracted from 4 cell lines, subsequently, RNAs were reverse transcribed into cDNA through reverse transcription Kit (Beyotime, https://www.beyotime.com/ ). Then the relative expression of these genes was detected using Real-Time Quantitative polymerase Chain Reaction (RT-qPCR).
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